FOOD AND DRUG ADMINISTRATION
DEPARTMENT OF HEALTH AND
HUMAN SERVICES
This
transcripts has not been edited or corrected, but appears as received from the
commercial transcribing service.
Accordingly, the food and Drug Administration makes no representation as
to its accuracy.
Meeting of:
TRANSMISSIBLE
SPONGIFORM ENCEPHALOPATHIES
ADVISORY COMMITTEE
September 18, 2006
Holiday Inn
Reported By:
CASET Associates
(703) 352-0091
TABLE
OF CONTENTS
Page
Administrative Remarks - Executive Secretary 1
Opening Remarks, Glenn Telling 8
Committee
Updates:
- US and Worldwide BSE 8
- vCJD epidemiology and transfusion transmission 26
- Draft Guidance for Industry: Amendment (donor 32
deferral for transfusion in
- Critical Factors Influencing Prion 39
Decontamination Using Sodium Hydroxide
- Human Prions: Clearance
and Plasma Lipoproteins 53
TOPIC
I: Experimental Clearance of
Transmissible
Spongiform Encephalopathy Infectivity
on
Plasma Derived FVIII Products 69
TSE Clearance studies for pdFVIII: Study Methods and 70
Clearance
Levels
Industry TSE Clearance Studies for pdFVIII 95
Open Public Hearing 116
- Statement by Dave Cavenaugh 118
Open Committee Discussion, Questions for the
Committee 122
Committee
Updates:
Status of FDA's Initiative on Communication of the 177
Potential
Exposure to vCJD Risk
Summary of WHO Consultation on Distribution of 184
Infectivity
in Tissues
Open Public Hearing 201
- Statement by Charles Sims 203
- Statement by Paul Brown 209
COMMITTEE
MEMBERS:
GLENN
C. TELLING, PhD, Chair.
VAL
D. BIAS, National
Hemophilia Foundation,
LYNN
H. CREEKMORE, DVM, USDA,
R.
NICK HOGAN, MD, PhD,
MO
D. SALMAN, DVM, PhD,
JAMES
J. SEJVAR, MD, CDC,
RONALD
S. BROOKMEYER, PhD,
BERNADINO
GHETTI, MD,
SUSAN
F. LEITMAN, MD, Department
of Transfusion Medicine, NIH,
JAMES
W. LILLARD, Jr., PhD,
MICHAEL
D. GESCHWIND, MD, PhD,
JAMES
MASTRIANNI, MD, PhD,
MARK
R. POWELL, PhD, USDA,
LAURA
K. MANUELIDIS, MD,
CONSUMER
REPRESENTATIVE:
NON-VOTING
INDUSTRY REPRESENTATIVE:
TARYN
ROGALSKI-SALTER, PhD, Merck & Company, Inc., PA
EXECUTIVE
SECRETARY:
WILLIAM
FREAS, PhD, CBER,
FDA,
COMMITTEE
MANAGEMENT SPECIALIST:
ROSANNA
L. HARVEY, CBER,
FDA,
P R
O C E E D I N G S (8:30 a.m.)
Agenda
Item: Administrative Remarks.
DR. FREAS: Good morning, and welcome to the 19th session
of the transmissible spongiform encephalopathy advisory committee. I am Bill Freas. I am the executive secretary
for today's meeting.
As announced in the Federal Register,
and amended in the Federal Register, today's meeting and tomorrow's meeting are
open to the public and the public is welcome to attend both days.
At this time, I would like to go
around the head table and introduce the committee members to the public. Would
the members please raise their hand when I call their name. I will be starting at the right side of the
room at the audience's right.
In the first chair is Dr. Sue Priola,
senior investigator, laboratory of persistent and viral diseases, Rocky
Mountain Laboratories.
The next chair is empty right now, but
it will soon be occupied by Dr. Michael Geschwind, assistant professor of
neurology,
The next chair is our consumer representative,
Ms. Florence Kranitz, president of the CJD Foundation,
The next chair is empty and that will
be soon occupied by a new committee member, Dr. Laura Manuelidis, professor and
head of neuropathology, Yale University School of Medicine.
Next we have David Gaylor, president
of Gaylor Associates,
Next we have our industry
representative, Dr. Taryn Rogalski-Salter. director of
Next we have Dr. Nick Hogan, associate
professor of ophthalmology, University of Texas Southwestern Medical School.
Next we have Dr. Mo Salman, professor
and director, animal population health institute,
Around at the head of he table is our
chair, Dr. Glenn Telling, associate professor, department of microbiology,
immunology and molecular genetics,
Next we have Ms. Jan Hamilton, advocacy
director, Hemophilia Foundation of America.
Next is another new member to this
committee, Dr. Mark Powell, risk scientist, office of risk assessment and cost
benefit analysis, U.S. Department of Agriculture.
Around he corner of the table we have
Dr. James Lillard, associate professor of microbiology, Morehouse School of
Medicine.
In the next chair we have Dr.
Next we have Dr. James Sejvar,
neuroepidemiologist, division of viral and rickettsial diseases, Centers for
Disease Control and Prevention.
Next we have Mr. Val Bias,
co-chairman, blood safety working group, National Hemophilia Foundation,
Next we have another new member, Dr.
Ronald Brookmeyer, professor, department of biostatistics, Bloomberg School of
Public Health,
Next, Dr. Susan Leitman, chief, blood
services section, department of transfusion medicine, National Institutes of
Health.
Next is another new member, Dr. James
Mastrianni, assistant professor of neurology,
Next we have Dr. Richard Colvin,
center for immunology and inflammatory diseases,
The empty chair will soon be filled by
Dr. Richard Johnson, professor of neurology,
Dr. Bernardino Ghetti could not be
with us at today's meeting. I would like to welcome everyone else and thank you
for coming.
Now I would like to read the conflict
of interest statement into the record. The Food and Drug Administration is
convening today's meeting of the transmissible spongiform encephalopathies
advisory committee under the Federal Advisory Committee Act of 1972.
All members of the committee are
special government employees or regular federal employees from other agencies,
and are subject to federal conflict of interest laws and regulations.
The following information on the
status of the committee's compliance with the federal conflict of interest
laws, including but not limited to, 18 US Code 208 and 21 US Code Section
355(n)(4) is being announced in today's meeting and will be part of a public
record.
FDA has determined that members of the
committee are in compliance with the federal ethics and conflict of interest
laws, including but not limited to 18 US Code section 208, and 21 US Code
section 355(n)(4).
Under 18 US Code 208, applicable to
all government agencies, and 21 US Code 355(n)(4), applicable to certain FDA
committees, congress has authorized FDA to grant waivers to special government
employees who have financial conflicts when determined by the agency's need for
that particular individual's services outweighs his or her potential conflict
of interest, section 208, and where participation is necessary to afford
essential expertise, section 355.
Members of the committee, including
consultants, appointed as temporary voting members, appointed as temporary
voting members, are special government employees or regular federal employees.
They have been screened for potential
conflicts of interest of their own as well as those imputed to them including
those of their employer, their spouse, minor child.
For the discussion topics, topic one,
which is experimental clearance of transmissible spongiform encephalopathy
infectivity in the plasma derived factor VIII products, and topic two, which is
possible criteria for approval of donor screening tests for vCJD, these
interests may include consulting, expert witness, testimony, contracts, grants,
CRADAs, teaching, speaking, writing, patents and royalties and primary
employment.
Today's agenda topics are considered
general matters discussions. In accordance with 18 US Code Section 208(b)(3),
general matters waivers have been granted to the following:
Drs. Ronald Brookmeyer, Michael
Geschwind, Bernardino Ghetti, James Lillard, Laura Manuelidis, and James
Mastrianni.
Previously approved waivers for Mr.
Val Bias, Dr. Lynn Creekmore, Dr. Nick Hogan, Ms.Florence Kranitz,
Dr. Glenn Telling, Dr. Mo Salman, are in effect for this meeting.
A copy of the written waiver statement
may be obtained by submitting a written request to the agency's freedom of
information office, Room 12-A-30, of the
Dr. Taryn Rogalski-Salter is serving
as the industry representative acting on behalf of all related industries and
is employed by Merck Laboratories.
Industry representatives are not special government employees and they
do not vote.
With regard to the FDA's guest
speakers, the agency has determined that information provided by these speakers
is essential.
The following information is made public
to allow the audience to objectively evaluate any presentations and comments
made by the speakers for topic one:
Dr. Lisa Ferguson is employed by USDA
in
In addition, there are regulated
industry and outside organization speakers at today's meeting making
presentations.
These speakers may have financial
conflicts of interest associated with their employer and with other regulated
firms.
These individuals who were invited
here to represent their companies, these individuals were not screened by FDA
for their conflicts of interest, since they are representing regulated
industry.
This conflict of interest statement
will be available for review at the registration table. We would like to remind
members that, if discussions involve any products or firms not already on the
agenda, for which they have a personal or imputed financial interest, they need
to exclude themselves from such involvement, and their exclusion will be noted
for the record.
FDA encourages all meeting
participants to advise the committee of any financial relationships that they
may have with firms that could be affected by the committee discussions.
So ends the reading of the conflict of
interest statement. Before I turn the microphone over to the chair, I would
like to ask, if you have a cell phone, please put it in the silent mode, so
that you won't disrupt those sitting next to you. Dr. Telling, I turn the
meeting over to you.
Agenda Item: Opening Remarks.
DR. TELLING: Thank you, Bill. I would
like to also welcome everybody here today. We have a full agenda so, without
further ado, I think we will get on to the committee updates. The first speaker
is going to be Dr. Ferguson, who will update us on
Agenda Item: Committee Updates. US and Worldwide BSE.
DR. FERGUSON: I am just going to go through. If you have my
handout, you see I have a whole bunch of slides, but I am going to rip through
things pretty fast.
They are pretty straightforward
slides, just to update what is happening in regard to BSE around the world, and
then finally, in the
Just a reminder for everybody, total
cases worldwide still is greater than 189,000 cases. Again, most of those, actually greater than
96 percent, have still occurred within the
So, when you still see all these
numbers of cases, the vast majority of that reflects what happened in the
If you are interested in current
totals, the OIE's web site actually has fairly good numbers, fairly updated
numbers, about all countries that report cases.
Let's start off and talk about what
has happened in the European Union. EU monitoring, since 2001, they have done
very intensive surveillance, mandated by legislation.
In 2005, they have recently released
their compiled report on all of the monitoring that went on in 2005, greater
than 10 million tests in all the 25 member countries, in cattle.
Of those, about 1.5 million are what
they call risk animals, which would be the same as our targeted population in
the United States, and 8.6 million approximately are animals either 24 or 30
months old at slaughter.
Of that, 561 positive cases, 448 of
those are in their risk or suspect animals, and 113 in the healthy slaughter
population.
Again, in 2005, same as in 2004, both
the number of cases and the overall prevalence in tested animals continues to
decrease. The number of cases decreased
by about 35 percent, overall prevalence by about 29 percent.
These reductions in the number of cases,
also the increasing age of positive cases -- if you read their report, there is
a lot of very good information about age of cases and how that progresses over
the years. I didn't include that there, because that would have made me go on
for far too long this morning.
Both of those do indicate success of
control measures in
The peak of exposure actually appears
pretty well defined in a few of the member states. This is the same as it was
last year, which is a good indication.
Either that is when the control
measures really started to kick in or, more important, it could also be an
indication of the increased surveillance that began in 2001. It is a little bit
early to tell.
They are also doing significant
monitoring in small ruminants, 614,000 tests in sheep and goats. Of that, 959
positives. Obviously, most of that is scrapie. They had no confirmed BSE cases
in small ruminants this year.
Just to show you what has happened
over the past five years within Europe, as you see, total number of positives
by year, and that is decreasing every year, the same thing here in the risk or
suspect animals versus healthy slaughter.
Those are very good indication that
the control measures that they have in place in Europe are working and are
doing the job that they are supposed to do.
Let's move to North America and a
brief update here on BSE in Canada. As a
reminder, Canada has been doing active surveillance, targeted surveillance, in
the population where they are most likely to find disease since 1991.
As everybody knows, in 2003, they
identified their first native case, in May of 2003. After that period, they
significantly increased their surveillance.
So, these are just their numbers, in
2003, about 5,700 samples, two positives. One was the one that we found here in
the United States in Washington State in December of 2003. So, increasing their surveillance again, with
no cases in 2004, two in 2005, and five to date in 2006.
They did provide a very detailed
epidemiological summary that was made public in January of this year. They are continuing that work and hopefully
will have some more updates out fairly soon.
What this summary shows, it really
talks about their geographic cluster theory, and the idea that the links
between rendering, feed production, livestock production, tend to occur in
clusters, in a fairly well defined geographic area. It is only logical, then, that that would be
where the disease could cycle, would be in that type of a cluster.
What this report shows, which went
through the first five cases, I believe, it does define links between most of
these cases and links in this cluster.
There are also linkages in the more
recent cases, again, to that same cluster and, as I mentioned earlier,
hopefully they will be putting out some more updates with as good epi work in
the near future.
To focus a bit on Japan, BSE was first
identified in Japan in late 2001. Actually, it was September 2001. They imposed
a feed ban, than, after that first diagnosis. So, the feed ban has only been in
place since 2001.
Here is the number of cases. It stays
about the same here until the past two years, where it has jumped up a bit.
One note about Japan. A lot of their
surveillance and the testing numbers that you see have been primarily in
clinically normal animals, or animals presented at slaughter.
It has only been since about 2004 that
they have really increased their focus on the targeted animals or the risk
population, as we would define it.
Now, let's move to the United States.
As everybody knows, we have been doing active surveillance in the United States
since 1990, and we are targeting the population where the disease is most
likely to be diagnosed. That is the most efficient way for us to conduct a
surveillance system.
The assumption is that if we can't
find disease in that population, then it is even more unlikely for us to find
it in the non-targeted population.
So, we can use the data that we get
from that targeted sampling to extrapolate information to the broader cattle
population.
Our targeted population has always
been, and continues to be, those animals that have some type of a clinical
abnormality that could even remotely be considered consistent with BSE.
So, these would be non-ambulatory
animals, dead stock, which are animals that die for some unexplained reason,
central nervous system cases, either called to our field people or on farms.
We work with veterinary diagnostic
labs, public health labs, for rabies negative animals, and then we also work
with our colleagues in FSIS, for those animals that are condemned on
ante-mortem inspections when presented to slaughter.
Everybody knows we ran an enhanced BSE
surveillance program that began in June 2004. Our initial intention was to run
that for 12 to 18 months, with the goal of getting as many samples from the
targeted population as we could.
We actually ran on a bit longer than
the 18 months and ran through August of 2006, greater than 785,000 tests during
that whole enhanced program. During that
time frame, there were two positives identified during that effort.
Just to show on a monthly basis what
we did in our enhanced program, as you can see, there is a little bit of a
cycle.
With the population that we are
sampling -- these are animals that are clinically abnormal in some way, and
with the facilities where we were collecting, these are animal disposal
facilities, rendering facilities, 3D, 4D, salvage slaughter plants.
Animals tend to get sicker, die, be
culled in the winter. So, we always had a little peak in the winter. I just
wanted to show folks, most folks don't quite understand exactly where we were
sampling and why that might occur.
Let me explain a bit about OIE
standards. These are the international animal health standards for BSE
surveillance.
This reflects changes to those
standards in May 2005. This is what we are using as our guidance for how we do
surveillance from here on out.
It is a weighted point system.
Previously it was a simple table that said if you had cattle population X, you
need to get Y number of samples.
Now it is an interesting system where
it recognizes that you are more likely to find the disease in certain
subpopulations.
So, you get more points for that
population where you are most likely to find the disease. We have four
surveillance streams.
They are clinical suspect, which would
be those animals with really pretty classic clinical signs of BSE, causality
slaughter -- these would be those animals -- these are European terms,
sometimes they fit with North American terms, sometimes they don't.
Casualty slaughter are those animals
that are clinically abnormal. They would be condemned on antemortem inspections.
So, these are those non-ambulatory
animals, could be the very weak, emaciated, thin, just some type of a subtle
abnormality.
Fallen stock are dead stock,
essentially, those animals that die for unknown reasons. Then, healthy
slaughter is pretty self explanatory.
I don't know if you can actually read
this. Hopefully you can. This shows the point system where it recognizes that
you are most likely to find disease here in a clinical suspect.
So, you get 750 points for a clinical
suspect between four and seven years old. That subpopulation is where you are
really most likely to find disease if it is present.
You get essentially limited points for
sampling in routine slaughter. So, what countries can do to use this table, you
can access whatever population you like to meet the standards, and then the
table says you need to get X number of points for a certain design prevalence,
300,000 points over a seven year period at a design prevalence of one in
100,000.
So, a country can then use this table
and figure out what type of samples in what population they want to sample to
reach those numbers of points.
So, you could sample a pretty small
number of clinical suspects and reach that point value, or design prevalence,
or you can sample a much higher number of routine slaughters. It recognizes that you can access both of
those populations. It is just how many samples you want to get.
We did a summary of not only our
enhanced surveillance program, but also what we have done for surveillance for
the past seven years. That was made public in April of this year.
Just to give you kind of a graphic
example of how to do this point system, these are the points that we obtained
in our surveillance for the past seven years.
So, close to three million OIE points
over the past seven years in the different surveillance streams. For those who
ask about this healthy slaughter one, knowing that we really are not sampling
healthy slaughter animals, this is a function of our data base.
Especially in some of these earlier
years, our data might be somewhat limited. If we could not pull out of the data
base a specific clinical sign, to assign this to one of the other surveillance
streams, by default it would go into the healthy slaughter for this calculation.
So, with that summary of data, not
only did we put out there just a raw data summary, we also did an estimate of
BSE prevalence in the United States.
We used two methods to do this. One is
the BsurvE model, which is a model developed by the Europeans with some input
from our colleagues down under in New Zealand.
It looks at what we know from the
epidemiology of the disease in Europe and factors in population data when
animals are most likely to leave the population.
It can be used to help a country set
up a surveillance system, can also be used to estimate prevalence.
We then also tweaked this model a bit
and came up with what we call the Baysian birth cohort model, which
incorporates what we would expect to see the effects of a feed ban, which the
Bsurv doesn't show, and it also sets up some linkages between birth cohorts.
We also did several sensitivity
analyses in this report, just to make sure that our assumptions weren't way off
base.
The overall conclusion was that the
BSE prevalence in the United States is very low, less than one infected animal
per one million adult cattle.
If you are interested in most likely
values, with the Bsurv model the most likely value was seven infected animals,
with the BBC model the most likely value was four.
With the sensitivity analyses, those
values ranged from one up to about 40, which all of those then led us to this
conclusion, pretty solid, that the prevalence is less than one infected animal
per one million cattle.
So, what are we doing now and where
are we going from there? We have used these same methods and have moved forward
into what we call ongoing surveillance.
We have been transitioning here since
the end of August. What we figure is about 40,000 samples per year, again,
still from this targeted population.
This will allow us to continue to
monitor the status of U.S. cattle and will allow us to detect prevalence if it
starts to increase.
We did this calculation, again, based
on our analysis of the enhanced surveillance data and using the Bsurv model.
We first of all looked at OIE
recommendations, which are at a design prevalence of one in 100,000. We wanted
to make that a bit more sensitive. We wanted to stick with one per one million.
So, we used the Bsurv model to estimate sample numbers and points.
Again, think of that basic premise
that I described for the OIE, with a different number of points for different
subpopulations.
With this, we need to get three
million analytical points over a seven year time frame. When we look at what we
did in enhanced, we averaged about 9.5 points per sample. So, we just divided
and that gets you to about 40,000 samples per year, assuming we will get the
same average points over a seven year time frame.
That is a very quick run through of
both an update of what is going on in the world, what the international
standards are, and where we are headed in the future. I think I have time for
questions.
DR. TELLING: Yes, you do. Thank you, Dr. Ferguson.
Are there any questions?
DR. GESCHWIND: Dr. Ferguson, when you were talking about the
Japanese cases, could you comment about the ages of the animals?
Before 2004, I believe they were
testing every cattle within a certain age range, and that they did find BSE
cattle among those that would not be identified with the current methods in the
United States. Can you comment on that?
DR. FERGUSON: I am not quite sure what you expect me to
comment on. I think as we all know Japan, in 2001, by their regulations,
required that every animal slaughtered, regardless of age, be tested for BSE.
They have recently changed that reg
slightly, and it is only animals 20 months of age and older be tested at
slaughter.
They did find two animals, a 21 month
old and a 23 month old -- unless my memory escapes me at this point in time --
young animals, apparently normal at slaughter.
They were positive on the screening
tests, negative on IHC, and then positive again with the way Japan has done the
western blot.
They have put those into mice to see
if there is transmission. To the best of my knowledge, those results aren't out
there yet, but there is no indication that they have gotten any signs of
disease in those mice.
Perhaps some of the researchers in
this group can clarify that, if I am mistaken on that point. That is the
situation in Japan.
DR. EPSTEIN: Lisa, my question is, does USDA have
information about food chain controls in non-U.S. countries? Are we in a
position to comment how adequate the food chain controls are from country to
country?
DR. FERGUSON: I can speak for AFIS per
se, since we are not the food safety group. That is really not part of the
information that we have.
Our colleagues in FSIS, through their
equivalency evaluations, work with certain countries, will have some
information on essentially the red meat inspection and control, similar to what
they would do in the United States. They would have that type of information. I
am not sure how much further in the food chain you need to go beyond that.
MS. KRANITZ: Dr. Ferguson, in the United Kingdom and Japan
they have found cases of BSE in animals that are not symptomatic. So, I would like to know why the USDA doesn't
consider random sampling of healthy stock.
DR. FERGUSON: I think it is shown in
that OIE table. We all recognize that you can pick up disease in animals before
they begin to show clinical signs.
It all comes down to what is the
purpose of your surveillance program and how do you best accomplish that
purpose.
In the United States, the purpose of
our surveillance program is animal health monitoring, to help us define either
the presence or the absence of disease in the U.S. cattle population.
The purpose is not to identify each
and every individual case of BSE that might be out there. In fact, that is an
impossibility to do with current test methods that are there.
So, we have chosen the most efficient
way by targeting that population where we are most likely to find disease if it
is present, to give us sufficient information to help us define the status of
the U.S. cattle population.
DR. MANUELIDIS: As a point of information, how many cows,
adult cows, are there in the United States, so that the 459,000 that were
tested is what percentage of the population versus Japan.
Then the second question is, the
Japanese found more cases. Would you sort of compare a little bit or say
something about the method of testing and the adequacy of the American testing
method in its generality as compared to the more extensive Japanese testing and
European testing.
DR. FERGUSON: Let me make sure I remember it. The first
question was about adult cattle populations. We estimate adult cattle
populations to be about 42 million currently.
I haven't done those numbers. I am not
going to stand up here and do math in my had and divide 759,000 over 42
million. I will let you guys do that, if you so choose.
As far as the adequacy of our
surveillance efforts in the United States compared to other countries, we feel
very comfortable and very solid with the information that we have obtained,
both over our enhanced efforts for the past two years, and all of the
surveillance that we have done prior to that.
The prevalence estimates that we have
done uses some very solid analytical methods, we believe, to come to the
conclusions that we have.
So, we feel like the surveillance that
we have done, targeted in the population that we have, is sufficient to help us
define what is going on in the United States.
DR. MANUELIDIS: I am really not trying to be difficult.
Perhaps you can't answer the question. I really wanted to know the specific
methods that you use and how they compare to the Japanese or the Europeans.
DR. FERGUSON: Sorry, I forgot that part of the question. I
assume you mean specific test methods.
DR. MANUELIDIS: Yes.
DR. FERGUSON: We are using -- actually, at this point in
time, I think everybody knows we are using the Biorad test for our screening.
Then, for confirmatory testing we will use both IHC and a western blot to help
confirm disease.
That is essentially the same as in
Europe. They have other rapid tests that are also available for use, not just
Biorad, but still confirmatory testing is with IHC and/or western blot. A
similar thing in Japan. They are also using Biorad, and confirmation is with
IHC and western blot.
DR. HOGAN: In terms of identifying your targeted
population, how are those animals being identified? Is it by government
employees or by industry, and what is your sense of the compliance rate?
DR. FERGUSON: We have had very good cooperation with the
industry over the past two years, actually since 1990, since we have been doing
surveillance.
As everybody knows, our surveillance
is not mandatory. We do have some regulatory authority to do that, but we have
chosen not to exercise it at this point in time. We have gotten where we are today with
cooperation with the industry.
In our enhanced program, since our
goal was to get as many of these samples as we can, it has not been an issue of
picking and choosing.
It is an issue of, okay, is this
animal old enough to meet our target, is it greater than 30 months of age, does
it meet this target, and is the sample of sufficient quality that you can test
it. If you are pouring the brain out, we
don't really want that.
So, that was sort of the criteria. We
have had AFIs personnel collecting samples, we have had state personnel
collecting samples. We have had contractors collecting samples, where we have
done the initial training, set them up on our data base, and go in and cross
check on them. So, it is a wide variety of folks who are collecting the samples
for us.
DR. TELLING: Okay, thank you, Dr. Ferguson. If there are
no further questions, I think we should move on. The second update is from Dr.
Scott, who will update us on variant CJD epidemiology and transfusion
transmission.
Agenda Item: vCJD Epidemiology and Transfusion
Transmission.
DR. SCOTT: This is going to be a brief
update of vCJD epidemiology and transfusion transmission cases. First, I am going to mention what the total
number of cases is worldwide of clinical diagnosed variant CJD.
The total number of cases right now,
as of August 2006, reported on the UK web site is 196. This is both deceased
and diagnosed and still alive.
These are the top three. In the United
Kingdom, there are 162 cases, in France 20, Republic of Ireland four, and then
there are a number of other countries that have had one to two cases each
reported, including the United States.
I want to point out that, in the case
of Italy and many of the cases in France -- about 19 -- and the Netherlands,
those patients in those particular countries had no significant travel outside
of their home countries.
So, in other words, it might be
speculated that these cases were acquired endogenously. Some of the other
countries had travel to the United Kingdom of less than six months, such as the
patient in Portugal and the patient in Spain.
The point I mean to make is that not all of these cases are directly
derived from visiting the United Kingdom.
The rate of variant CJD deaths has
been declining in the United Kingdom over the past several years. This is the
number of deaths from definite and probable variant CJD reported in the United
Kingdom.
I am showing you from 2000 to the
present, but remember that the first case was published in 1995 and probably
developed symptoms in 1994.
I am beginning here with the peak year
where there were 28 deaths from variant CJD in the United Kingdom. As you can
see, while the years go by, you get a decline in the number of cases. So, in
2006, there were three deaths reported.
I want to point out that, in the
United Kingdom right now, there are six patients right now still living with
this disease. So, it doesn't look as if
we are going to have a large number, as we did in the year 2000.
There have been three reports of
transfusion transmission of variant CJD in recipients of non-leukoreduced red
cell concentrates from donors who subsequently, post-donation, developed
variant CJD. They were healthy at the time of donation.
Two of these cases had already been
reported at the time of the last advisory committee meeting, but the third case
was reported in February of 2006.
Two of these cases were recipients
that developed clinical variant CJD. The donors to these patients developed
their disease about 18 to 42 months after they donated blood.
The recipients developed symptoms of
variant CJD six-and-a-half to eight years after receiving the transfusion from
these donors.
The new case had a donor who developed
clinical variant CJD 18 to 20 months after he or she donated, and the recipient
developed variant CJD eight years after receiving that donation.
In addition there was one infected
asymptomatic recipient of blood from a patient that developed variant CJD. This
person died of an unrelated illness five years post-transfusion but, at
autopsy, the variant CJD associated PRP protein was found in the spleen and
lymph nodes of this person.
The other thing that makes this case
unusual, besides being diagnosed when asymptomatic, is that they were
heterozygous for methionine and valine at PRP codon 129.
So, this is a genotype of the prion
protein that previously had not been reported in people with clinical variant
CJD.
This was the first and the other two
types are MM, which are all the clinical cases reported so far, and VV, and we
will get to that in a minute.
To continue on the same theme, I am
showing you an update or new information that has recently come out concerning
the study by Hilton et al that was published in 2004.
This was a United Kingdom tissue
survey where anonymized tonsil and appendix samples were taken from subjects
that had undergone surgery between 1996 and 1999 in the United Kingdom.
The samples that were studied were
from patients aged 20 to 29. Very interesting and important, three out of the
12,674 samples that were deemed adequate for study were positive, suggesting
one in 4,225 people in this age group might actually be infected with variant CJD. All of the positive samples did come from
appendices.
Now, what is new about this is that
prion protein genotyping was done on two of these samples. In this first sample, there was not enough to
do genotype testing, but the tissue was taken and used in a transmission study
into mice, and those results are pending. We don't know what has happened to
those mice just yet.
In the second subject sampled, the
genotype was found to be valine homozygous. So, this was the first report of an
infection in a valine homozygous person.
The second was also a valine homozygous individual.
So, to summarize, variant CJD clinical
cases are declining in the United Kingdom. We have had three transfusion
transmission infections reported in the United Kingdom, one fairly recently.
I would just like to point out that,
out of the 18 identified living recipients of blood from people who came down
with variant CJD, recipients that have survived at least five years
post-transfusion, now three out of 18 of these people have developed vCJD
infection. Two of those are clinical and, as I showed you, one of them was
preclinical or subclinical at the time of death.
This implies a fairly efficient
transmission by blood. This amounts to about 17 percent. Also, we now know that
all three prion protein genotypes at codon 129, the MM, the MV and the VV, are
susceptible to infection.
What we don't know is whether people
with this genotype ever develop clinical illness. This brings up the continued possibility that
there are silent and asymptomatic infections that may never become symptomatic,
but may pose a risk of iatrogenic transmission to others.
In particular, we are concerned with
blood and plasma, but there are other iatrogenic transmissions to be considered
as well. That is all for my update.
Thank you all for your attention.
DR. TELLING: Thank you, Dr. Scott. Any
questions of clarification for Dr. Scott?
DR. GESCHWIND: Dr. Scott, just regarding the Saudi Arabia
case, as I recall, when that was originally presented at the academy meeting,
that patient had lived in the United States for, I believe, greater than six
months and, on the way to the United States had spent the night in London on
the way there. Any thoughts about that, in terms of the risk of transmission?
DR. SCOTT: I think that is somewhat similar to the
Japanese case, where that person spent 24 days. This has been reported so far
in two different ways that I have seen.
One is the WHO report dating from
June, where they state that only in France, Italy and the Netherlands did the
people have no significant travel outside their home countries.
In the United Kingdom, it is reported
differently, in that they looked at cumulative residence in the United Kingdom
of greater than six months, how many people in the other countries had that
kind of residence in the United Kingdom.
In Saudi Arabia, they report zero. So,
obviously, that is a person that could have been there for a night or three
months and wouldn't have been counted in the UK way of tabulating things.
I think the question does always
become, was this endogenously acquired or acquired in some other country, and
it probably isn't possible to answer that for certain. It may be that a single exposure to high
titer BSE could infer infection.
DR. COLVIN: In the case of the UK tissue survey, out of
the patients that were surveyed, that 12,674, was it known if those people, for
one thing, had had any blood transfusions or, secondly, if any of them had been
users of any kind of plasma or plasma-derived products.
DR. SCOTT: That is a very good question. In order to get
the study accomplished, they had to completely anonymize the samples. So, those
people will never be identified and we don't have any information about them.
DR. TELLING: Any other questions for Dr. Scott? If not, thank you very much. Our next speaker is Dr. Williams. He is going
to talk about a draft guidance for industry, an amendment. He is going to be
talking about a donor deferral for transmission in France since 1980.
Agenda Item: Draft Guidance for Industry: Amendment.
DR. WILLIAMS: Good morning. I am going to present a very
brief update on recent draft guidance issued by FDA pertaining to deferral of
donors with a history of transfusion in France since 1980.
The current recommendations are for
vCJD related donor referrals, as have been seen by this committee many times,
but I wanted to runt through them quickly just for the new members of the
committee.
Most of these, or all of these, are
captured in guidance to industry published in January 2002. Deferrals include
residents with travel of greater than or equal to three months in the United
Kingdom from 1980 to 1996, residents with travel of five years or greater in
Europe for the same period of time.
For donors of source plasma, this
criteria applies only to France, which is considered to have five to 10 percent
consumption of UK Beef, and therefore be at proportionately higher risk
compared with the rest of Europe.
Combined with the presumed prion
production reduction in the course of fractionation, this deferral was modified
specific for plasma donors.
In addition, donors who spent greater
than or equal to six months on U.S. military bases in Europe between 1980 or
1990, or 1980 to 1996 respectively for regions in the north and the south, are
deferred.
This is based on importation of UK
beef into U.S. commissaries, and this differed between the northern and
southern bases during this period of time.
The guidance also defers donors for
history of transfusion in the United Kingdom from 1980 to the present and for
receipt of bovine insulin sourced in the United Kingdom after 1980.
At the meeting of this committee on
October 14, 2004, the committee reviewed current FDA regulations regarding vCJD
related donor eligibility.
After considerable discussion, they
did not make recommendations for further FDA actions to protect the blood
supply.
However, there were discussions at the
meeting concerning the predictive value of donor questions that were used to
exclude TSE risk and just how effective the questions were, as well as the
feasibility of deferral for history of transfusion outside of the United
Kingdom, but no specific recommendations were made at that meeting.
Subsequently, at the February 8, 2005
meeting of the committee, FDA brought the issue back for consideration, based
largely on several recent observations at that time.
At that time there were two observed
variant CJD transmissions associated with transfusion and it was recognized
that two recent variant CJD cases observed in France had had a relatively large
number of prior blood donations.
That is not necessarily a scientific
rationale for considering the issue, but it really did raise the visibility of
the fact that any potential patient could be a blood donor.
At the time, as well, there were some
actions in Europe where donors were deferred for any previous transfusion.
In france, this had been in place for
some time since 1998, in The Netherlands deferral was implemented for any
history of transfusion in 2004, and in the United Kingdom this took place in
2005.
Also discussed at that meeting was the
potential impact of any increase of donor deferral for history of transfusion.
The history of transfusion deferral
for the United Kingdom had already been accomplished. The calculations for this
are reflected in the transcript, but were estimated to be about two per 10,000
donors.
Computed proportionally, the estimated
loss for donor who had a history of transfusion in France was figured to be 1.4
per 10,000 donors.
Also, under discussion at that
meeting, was the potential for deferring any donor who had a transfusion
anywhere in Europe.
When considering this excluding the
United Kingdom, that would add another three per 10,000 donors. There were no data available regarding source
plasma donors and their history of transfusion or travel, but simply due to the
younger age group of the source plasma donor, this would be expected to be
somewhat less.
In the TSEAC deliberations at that
February 8 meeting, TSEAC recommended deferral of blood donors transfused in
France since 1980 by a vote of 12 for, three against, and one abstention.
However, the committee did not
recommend deferral of blood donors transfused elsewhere in Europe since 1980 by
a vote of zero to 15 against and one abstention.
By a somewhat mixed vote, the
committee also did not recommend deferral of plasma donors transfused in
France. That vote was five for, seven against and seven abstention, or other
European countries, with a unanimous vote of 16 against.
In issuing its draft guidance for
industry, FDA is basing the guidance on the rationale of being prudent
preventive measures to help prevent or reduce the risk of vCJD transmission by
transfusion.
These factors are the relative
likelihood of dietary BSE exposure in France - and this has been an
underpinning of any of the discussions that have been held through the years
about potential donor exposure.
At the time of assembling the
guidance, there had been three presumptive cases of variant CJD transmission by
transfusion. So, this was no longer a theoretical possibility.
There were 14 definite or probable
vCJD cases observed in France. It is now observed that the variant CJD
incubation period may be as long or longer than 12 years, and asymptomatic
prionemia may be over three years prior to the expression of illness in an
infected donor.
With respect to plasma donations,
experimental studies of prion reduction and fractionated plasma are reassuring.
However, not all fractionated products
have been studied and observations do not necessarily reflect the blood form of
the variant CJD agent.
So, the guidance itself is published
as a draft amendment to the January 2002 guidance entitled, Donor Deferral for
Transfusion in France since 1980.
FDA recommends deferral of donors who
have received a transfusion of blood or blood components in France since 1980.
This applies to whole blood and blood
components intended for transfusion, as well as blood components intended for
further manufacturing into injectable products including recovered plasma,
source leukocytes and source plasma.
The implementation target for this is
within six months of publication of the final guidance and the mechanism that
will be used.
This amendment is published as draft,
but the 2002 guidance will be updated and published in final to incorporate
this as final guidance. Thank you.
DR. TELLING: Thank you, Dr. Williams. Question?
DR. HAMILTON: Has there been any consideration in the
Untied States of the areas of the country that the people tend to eat brain
matter and offal, consuming that? Has
there been any consideration of that?
DR. WILLIAMS: There has been consideration of it. In fact,
the NHLBI sponsored red study actually did a survey of consumption of brain
matter and had some preliminary data on that.
I think this has been an area of
consideration but I think to date the scientific evidence supporting this as a
potential factor in transmission of disease hasn't been strong enough to
support this as a deferral.
DR. TELLING: Thank you very much. If there are no further
questions we can move on. Dr. Cai from Telecris Biotherapeutics, will discuss
some critical factors influencing prion decontamination using sodium hydroxide.
This is a PTTA collaborative study.
Agenda Item: Critical Factors Influencing Prion
Decontamination Using Sodium Hydroxide.
DR. CAI: Good morning.
Thank you very much for inviting me. On behalf of the Plasma
Therapeutics Protein Association, I would like to present the results of a
collaborative study designed to understand the critical factors influencing
prion decontamination using sodium hydroxide.
This work was primarily done at
Telecris Therapeutics. It used to be Bayer.
Some of the work was done at Bioreliance.
So, we are all working together to
establish a systematic, comprehensive approach to minimize the risks associated
with potential SE transmission.
So, as the first line of defense, we
have donor deferral and material control.
As a second line of defense we have the capacity of the manufacturing
processes to remove or clear prions.
In supplement to that capacity, we
have cleaning and sanitization procedures. If those procedures can inactive
prions, that would add additional benefit in terms of risk reduction. So, this
is my focus of the talk today.
So, speaking of prion inactivation, we
know that a prion is very resistant, very difficult to inactivate, using
conventional viral inactivation methods, simply because, on the one hand, it is
a nuclear acid base to viruses and we are dealing with protein prion infectious
materials.
However, the prion has its own
vulnerability, and it has its own weaknesses, because proteins can be unfolded
under many circumstances.
Over the time, researchers have
developed many methods to inactivate prions, including enzymatic digestions
coupled with detergent treatment, and also acid or strong base treatment, or
also some other chemical reactions such as titanium dioxide, photocatalytic
inactivation.
Let's not forget our old friend, which
is the strong base, which has been used widely in the industry to clean
equipment, which includes potassium hydroxide and sodium hydroxide.
Those are readily available and
inexpensive, rapid, ineffective and comparable with most of the major
equipment, like stainless steel equipment, although it is not very comparable
with silicon based materials. Those can be treated or disposable.
So, actually, over the time,
researchers performance many studies on sodium hydroxide in terms of
inactivation of prions, all the way back to the 1980s.
They used various spiking material or
model prion agents for either rodents or humans, and they examined various
concentrations, various temperatures, as well as various incubation times in
terms of treatments. Of course, you got
various results as well.
Then this is a reduction factor of the
output prion titer relative to the input after specific treatments. You can see
there are various reduction factors based on the conditions.
However, if you look closely, whenever
there is a presence of detergents, there is a good reduction. So, please keep
this in mind, and this is very relevant to our discussion.
Secondary, you can also observe that
among these reduction factors, you know, most of them are quite effective.
However, not all of them give a
complete inactivation. In other words, there is still residual infectivity or
prions remaining after the treatments. So,
the question is why.
That is quite consistent to what we
have observed during the early stage of our study, the Bioreliance. In this
case used scrapie brain homogenate, which contains hamster prions at one
percent, and mixed with sodium hydroxide at .1 molar, incubated at 18 degrees.
The top panel is the titration of the
input material. So, you can see quite significant amounts of prions gives us --
this is a half log dilution.
After the treatment you can see the
majority of the signals -- the signal strength is significantly reduced.
However, there is still remaining signal.
So, there are two questions here. One
is, what is behind this dramatic reduction after the treatment. The second
question is, what is the nature of this residual signal.
So, we set to address these questions
by designing experiments that would mix the purified scrapie brain homogenate
with sodium hydroxide, and incubate it with or without two percent sarkosyl,
which is a detergent, after incubation at the various temperatures and the
various time periods.
Then the sample is withdrawn and
neutralized and treated with proteinase K in order to detect the pathogenesis
conformation, as is run by electrophoresis and detected by western blot to see
the signal strength of the prions.
Now, what we observed was that if you
have detergents in the sample, then the residual signals can be eliminated to
below the detection limit of western blot.
On the upper left panel at four
degrees, you have this much of input material. In the absence of sarkosyl,
after 60 minutes, we observed about three logs of reduction with a residual
signal.
When the temperature is elevated, then
the reduction is slightly increased. However, in the presence of sarkosyl, at
15 minutes, the reduction is increased and, at 60 minutes, the reduction is
more than 4.6 logs. So, it reduced below the detection limit. At the elevated temperature, this
disappearance occurs earlier.
So, we know that a detergent mainly
affects the lipid composition or the disrupted structure of the lipids and
detergent has very little effect on the overall structure of proteins.
So, it is highly possible that, in the
sample, there are two subpopulations of prions. One is protective by lipid
components and the other is protein alone.
So, this one is protective against,
not accessible, by the sodium hydroxide. So, after the treatments and when you
use proteinase K to probe the structure, obviously this structure is not going
to be digested, resulting in a remaining signal.
Now, in the presence of detergents,
the protection is removed and the entire population is vulnerable to sodium
hydroxide or extreme pH. Then, when you
use this probe to probe the structure, then the structure is no longer there.
Now, prions are about protein folding
and miscoding. So, it can exist in a
normal conformation with alpha helixes and exposed epitopes. Those epitopes can
be accessed by antibodies as well as proteinases.
It can be mis-folded into this
pathogenic form. So, in this case, the structure is mainly beta sheet and the
epitopes, some of the epitopes, are buried and no longer accessible by the
antibody or proteinases.
Then in order to inactivate this
moiety we need to somehow unfold it or degrade it. So, this is the place, I
think, that is for the prion activation.
So, this experiment was designed to
further analyze the conformational change behind the prion inactivation or the
structural change of prion proteins upon the incubation with sodium hydroxide.
In panel A, which is in the absence of
detergent, and absence of proteinase K treatment, the trace buffer saline is a
control.
It gives an input, the titer of input
sample, and sodium hydroxide treatment you can see pretty much remaining, the
signals are pretty much remaining the same, with very little reduction.
That tells us the peptide chain
backbone is pretty much preserved after the treatment. However, the
conformation is no longer there because in the panel B, once you use proteinase
K to probe this structure, the structure is obviously greatly damaged, with a
big reduction in terms of titer.
However, there is a little residual signal again.
Now, in the presence of detergents,
again, the protection was supposed to be removed after sodium hydroxide
treatment.
There is 90 percent of the signal that
was reduced in the absence of proteinase K, which tells us actually the peptide
chain in this case, or the side chains of the epitope are damaged.
In the presence of both detergents and
proteinase K we can see the signal disappeared completely using this assay
because the structure is unfolded and it was digested by proteinase.
To further support these observations
we conducted immunoprecipitation assays, tried to demonstrate that the sodium
hydroxide unfolds the prions.
So, in the normal conformation this
epitope is exposed to the solvent, accessible to antibody. If you have antibody
beads, then you can immunoprecipitate the structure.
That is what we observed for this
amount of input material in the buffer control, or preneutralized sodium
hydroxide, which says that there is the same amount of solutes of the base, but
it was neutralized before.
So, the sample does not experience
extreme pH. So, in both cases you can see a good recovery of the signal by
immunoprecipitation.
Now, it is totally opposite to the
pathogenic conformation, where the epitopes are buried and you will not be able
to precipitate it. In this case you see
no or very low signal.
Now, treatment with sodium hydroxide,
it unfolded this structure and made this epitope accessible. So, you can see
the immunoprecipitation.
This is pretty much, the overall
consequence, is pretty much similar to what guanidine kinase does, which is
chemotropic agent, unfolds the prion, which is used as a control in this case.
So, in summary, we think the critical
effects influencing decontamination using sodium hydroxides include, of course,
the concentration of the agents and the presence of detergents, and temperature
and time also contributes, to some extent. So, overall, the sodium hydroxide
works by unfolding and degrading the structure of the prion.
So, I would like to thank people who
contributed to this study, especially the PPTA collaborators. Also, the
experimental work was done by Dr. Pat Bauman and her research team.
I would also like to thank
contributes, former PPTA members, as well as others who contributed to this
study. Thanks.
DR. TELLING: Thanks, Dr. Cai. Are there any questions?
DR. SALMAN: Can you comment on what type of media you
used for the prion? What type of vehicle
do you have it in?
DR. CAI: You mean the spiking material?
DR. SALMAN: Yes.
DR. CAI: That was clarified brain homogenate from
masters with the 263K strain.
DR. SALMAN: Have you tried to see if there is any type of
effect of the organic matter on the decontamination?
DR. CAI: We haven't specifically examined it in that
respect.
DR. SALMAN: I have another question. What is the reason
to decide, as far as the maximum temperature of 18 centigrade?
DR. CAI: We chose several temperatures, such as four
degrees, 18 degrees. Actually, those are
conservative. So, we tried to model production processes, manufacturing
processes.
During manufacturing, the cleaning
procedures vary depending on manufacturer, such as whether it is upstream, is
it downstream, whether it is -- the condition of the equipment.
So, there are hundreds of standard
operation procedures for each manufacturer to define the specific procedures
for cleaning.
All those procedures are validated
based on a validation package, you know, according to common practice, where
you use many measurements to determine how much residual protein is remaining.
Often we use total carbon
measurements. So, if the total carbon measure is under a certain level, then
you are confident there is no residual, or small amounts of residuals,
remaining.
In that regard, back to your question,
it is an organic compound and those effects are probably very limited.
DR. MANUELIDIS: I would like to sort of clarify something
here, to make sure that in the rest of the meeting this is clarified by the
speakers.
The question is, did you ever
inoculate any of this material to see how infectious it was? You are making an assumption about
inactivation of abnormal prion protein and infectivity, which other types of
studies -- there are many numerous studies including heat inactivation,
guanidinium, et cetera, where the correlation is not there.
So, I would really like to know, did
you do any infectivity studies? I think
it can be misleading to sort of say that we have inactivated this as a
titer. Titer usually refers to
infectious titer as measured biologically.
DR. CAI: We did do infectivity study, although the
data is not shown here. We observed quite good correlation between the
inactivation measured by western blot as well as hamster bioassay.
In addition, previous studies also
demonstrated, as listed here -- a lot of them were done by using a bioassay,
for example, infectivity as it was labeled as a green star here. Many of those
cases are done by bioassay. So, they have quite good correlation.
DR. SEJVAR: Just to clarify, the conditions that you have
been describing would be compatible with real life experience, in other words,
actual decontamination of, say, surgical instruments, et cetera.
DR. CAI: Actually, there is some subtle difference
between surgical instruments and the manufacturing processes for plasma
products.
A surgical instrument is in direct
contact with central neural systems and often has a much, much higher degree of
protein binding to those instruments.
The manufacturing equipment, on the
opposite side, has -- it is rigorously cleaned, of course, but if you think
about if there is any donation got into the manufacturing side, the infectious
titers could be very, very low.
So, it is kind of a different scenario
but the general approach should be applicable to both. A lot of the surgical
instruments are also treated with sodium hydroxide.
DR. SEJVAR: But you are talking about specifically plasma
products, et cetera?
DR. CAI: Right.
DR. MASTRIANNI: In your experiments you used sarkosyl at two
percent. I am wondering if you did a titration curve to see if there was a dose
response that correlated with increasing levels of detergent to show a decrease
in signal of western blot.
DR. CAI: That is a good point. Although we didn't
titrate it, we did a spot test. You do need a certain amount, one percent, two
percent, but if you go down to .1 percent, the effect will be reduced.
DR. COLVIN: As opposed to the indirect
method of looking at the structure of the prion protein through either
proteinase K susceptibility or through the western blot through antibody
affinity, did you look at any direct measures of conformational change, such as
using circular dichroism, NMR, spectroscopy, something that would show there
has been a change, or even differential centrifugation of the products?
DR. CAI: Limited by our methods, we were unable to use
a lot of physical means. We did these studies primarily in the BSL-2 lab in our
setting, which is set up for pathogen research.
So, we don't have extensive physical characterization testing available.
DR. GESCHWIND: Related to those last
two questions, one is, there is clearly a difference in inactivating human CJD
as in animal, as shown in the Peretz paper, Journal of Virology that came out
recently, where human prions were found to be 100,000 times more difficult to
inactivate than in animals, hamster 263 prions.
I think that is an important point to
consider, that the only effective way of testing really should be -- I think
this is an important point for the committee to consider for the next two days,
is that really the human prions are going to be different than animal prions
and we have to realize that.
Then the second issue is, is there any
equipment in the processing for the plasma that would be exposed to metal, as
clearly there is a difference between brain homogenate and testing in which
they have looked at small pieces of metal put into the brain.
So, brain homogenate has always been
easier to inactivate than the steel rod method. I am just wondering if there is
any possible exposure to metal in particular during any of the processing.
DR. CAI: Yes, the first question about Dr. David
Peretz' paper, I guess using acid in combination with detergent to inactivate
prions. So, they compared between
hamster and human materials and there was a big difference.
I would like to point out in that case
it is weak acid at a pH 3, 4. In our case, sodium hydroxide or potassium
hydroxide is a strong base. It is a strong electrolyte. So, they are very, very
different in their nature.
In addition, the previous studies
demonstrated by almost 10 groups using the sodium hydroxide treatments with the
absence of detergents, they consistently demonstrated significant removal or
reduction using various strains of material, including rodents and humans.
So, the second question is whether the
manufacturing process has metal components in the equipment. The answer is yes.
Of course we use a lot of metal equipment, including stainless steel, all of
that.
Several groups, including Dr. Safar's
group, they use metal instruments into rodents to detect the prion infection.
That is a very good approach and gives us a better understanding.
The study we did, you know, they were
designed to address in general those conformational changes, and to understand
the significance of a residual signal, how to remove residual signal. Those
studies should be complementary, I think.
DR. TELLING: Thank you. We need to move on. The final
speaker in this update section is Dr. Safar from the University of California,
San Francisco, who will be talking about human prions clearance in plasma
lipoproteins.
Agenda Item: Human Prions: Clearance and Plasma
Lipoproteins.
DR. SAFAR: First of all, I would like to thank the
committee for this opportunity to present some new data that we think are very
relevant to the task of this committee.
I think that fundamental issues facing
the prion research, I have selected those that I think are very relevant for
this meeting and for the present agenda.
I think that the three in the square
are interrelated. The pathogenesis of the prion diseases is important to understand
and to plan the most effective therapeutics which would halt the prion
formation and remove existing prions.
The condition for that is to have a
very sensitive presymptomatic diagnostic test. If we would initiate any therapy
late in the symptomatic stage of disease, there is very little hope for the
recovery. The situation is very similar
to Alzheimer's disease.
This is a table which I put in. It is
very complex, but I think that it is really important to realize the progress
of the field in the last few years.
There were originally described two
entities related to the prion diseases, normal PRP, cellular form of the PRP
protein and the resistant form of PRP, PRPSC, which is infectious.
We found that there are very similar
species, which actually in many cases is dominating disease, we called protease
sensitive form of PRPSC.
They have different conformations. The
PRPC has exposed most of the epitopes against monoclonal antibodies, where PRPSC,
both S and R forms, those epitopes -19 and -125 are already buried.
The secondary structure of PRPCs is 40
percent helix. RPRPSC is 40 percent beta helix. We don't know the
conformation of the PRPSC.
Quaternary structure of PRPSC
is a monomer PRPSC or oligomers, and $PRPSC can
polymerize into analoid-like rust.
Standard PK destroys both PRPC and
SPRPSC, but leaves behind a proteolytic fragment of PRPSC
which we call PRP22-30 by molecular weight.
Current PK, which we found very
simply, hydrolyzes selectively PRPC and leaves behind a proteolytic fragment which
is typical for SPRPSC. So,
that is the first really direct evidence that this comes separately from what
the RPRPSC comes from.
Another way to separate PRPC and PRPSC,
both S and R, are polyoxometalate, polyoxometalate precipitation.
Non-denaturing detergents are solubilizing PRPC, they have mixed effect on the
SPRPSC, they don't solubilize RPRPSC.
Infectivity, normal protein is
non-infectious, of course, and the RPRPSC is infectious. Levels
during infection, there is no change, no up-regulation of PRPC. SPRPSC
and RPRPSC are in equilibrium, which is typical for different prion
strains.
In RML infected mouse, the clearance
half time for the SPRPSC and RPRPSC is 1.5 days. I showed this slide because I think it is
becoming increasingly important.
There is a large percentage of the
sheep scrapie which carries selectively the SPRPSC forms, and
wouldn't be detected without the availability to detect this PRPSC. We
saw it in Norway cases and more and more cases in Europe.
There is a growing number of human CJD
cases, and they were presented last week in San Francisco by Luigi Gambatti(?)
from his CJD surveillance collection.
He estimates that it may be up to 14 or
15 persons which display selectively SPRPSC and practically no
detected RPRPSC proteins.
So, this is becoming very important
for two reasons. First, practical
detection and identification of the prion disease. Second, in a theoretical
sense, how is it related to the disease and how important is it in
pathogenesis.
The direct PRP protein with proteases,
we designed 10 years ago the protocol which avoids proteinase K. It is called
conformation dependent immunoassay, and it recognizes antibodies which are
exposed in PRPC and hidden in PRPSC.
This is the beta helix of PRPSC, helix A and C, which are
still remaining there.
If you test simultaneously one sample
which is native and the second of which is denatured after denaturation with
sodium hydrochloride, you compare the signal.
If you don't see any increase in the
signal, you know that they are PRCP proteins, or if there is a very small
increase, you can account for it by establishing for the size of cattle.
If there is an increase in the signal
after denaturation, you know that you have a certain percentage of PRPSC
in the original sample, which had hidden epitopes. that is a quantitative
parameter indicating the presence of PRPSC protein.
We weren't very happy with the
sensitivity. So, we are looking for the compounds which would selectively
precipitate PRPSC and leave behind PRPC. One of those compounds was
the keggin structure of polyoxometalate, where the phosphate is in, and they
are coordinating the constant oxide of the hyderons around.
There is a misconception that it is
some small cell. The polyoxometalates are actually very large. The monomer of
PRP structure, monomer of PRPC is about 1.8 nanometers. The polyoxometalate in
this case, kaggin structure, is about one nanometer large.
So, those are very large compounds
which can be synthesized in a way which modulates either size, shape or charge.
We found out that some of the
polyoxometalates are efficient in aggregating PRPSC, the S and R
forms, and large polyoxometalates actually have a positive effect. They
dissociate PRPSC proteins.
This dual effect is still not
understood exactly at the molecular basis. It is definitely related to the
charge and size polyoxometalate.
So, kaggin structure, small
polyoxometalate cyclates facilitate prion formation and decrease cell growth
from prions, and the large polyoxometalate have the opposite effect. They
dissociate PRP C protein and they make smaller complexes.
Our studies of SVRPLC proteins, the
protease sensitive form, was initiated with generating biogenic systems to
regulate the PRPC level of expression.
So, we could shut down PRPC and look
what happens to PRPC proteins, both the S and the PRPSC. The first
surprise came following the incubation time.
When you shut down PRPC expression and
then follow the incubation time of the animals, those which express
downregulated PRPC from 100 percent to about five percent of residual
expression, extended their incubation time by about three-fold.
So, that was a really amazing result.
We didn't expect it because we were afraid that the small leak we have in the
background would inevitably lead to very small changes in incubation time.
When we measure the PRPC protein
prions, we found that the PRPC has a half time of about 18 hours, PRPSC
has a half time of about 36 hours, measured by both CBI and western blots.
So, that was really an amazing
finding, indicating that this is a very powerful mechanism in the brain,
physiological mechanism, which is able to clear prions in one and a half day,
and practically 50 percent of already formed prions.
It is apparently related also to the
strain. When we compare CO1V, an animal strain, CO1V, which has a slightly
extended incubation time, it cleared about twice as more slowly. Also, the accumulation was slower.
So, there is an interrelationship
between the stability of the prions for prion strains, incubation time, the
accumulation rate and clearance rate. Those functions are strain specific.
If you look at the pathology of the
animals where the PRPC was likely to be expressed during the incubation time of
prions, we see large deposits of PRPSC proteins.
If you look at animals which were
inoculated and then, after 98 days, which is about two thirds, we shut down
PRPC, we see how clean those brains are.
There are only some deposits in the
corpus callosum in the white matter. Most of the cellular areas are completely
clean. Other deposits are around the
vessels.
This slide, I think, is really optimistic.
It has got a therapeutic approach. If we would be able to down-regulate PRPC,
you would effectively cure the disease because the brain has a very powerful
clearance mechanism for clearance.
Additionally, the therapeutic level,
we know now that it is possible that the prions are continuously synthesized at
the low level and that the brain has -- that they have some physiological
function in the brain, and that the brain has, at the same time, a very strong
clearance mechanism, which is how you get rid of them.
So, where do prions go? We know from other experiments, when you
inoculate directly prions into brain, 99 percent are lost within the first 24
hours.
So, there is massive outflux of prions
from the brain, and obviously the target in this case has got to be the first
circulating blot and cerebral spinal fluid.
There is no question that there is
infectivity in the blood, and there are many studies indicating them. The issue
in this case is which compartment of blood it is. Is it out of blood or plasma
or both.
So, we established a system where we
looked really blindly in both plasma and white blood cell compartments. White
blood cells are sorted by facs, flow cell activated cell sorting, and by myelin
Bs, and we have tested up to now granulocytes, monocytes, CD4, T cells, B
cells, circulating dendritic cells from different animals and also from CJD
patients.
The results are -- the results really
increased in plasma. We started to supply them with polyoxometalates.
Polyoxometalates have not only very specific precipitation capability of
prions, but they also precipitate lipoproteins.
They have a still not fully understood affinity to the lipoproteins.
So, we decided to test where the
prions would go in the human plasma by using polyoxometalate fractionation. By
increasing concentration of polyoxometalates, you can supply plasma into the
LDL particles, lipoprotein particles, immunoglobulins, HDL and other plasma
components.
When we spike the plasma with prions
from brain, sporadic CJD prions -- so, this is a homologous system, plasma,
human plasma, and sporadic CJD and one case of CJD.
We found that all the prions, by
western blots, and by CDI, were fractionated or precipitated into the VLDL or
LDL fraction of the lipoproteins.
It is not actually so surprising. The
PRP, prion PRP has a very high affinity for cholesterol. It is very difficult
to separate them.
So, the lipoprotein particles that are
about 60 percent of cholesterol and phospholipids, and about 30 percent
hydrophilic proteins. So, the fact that they have affinity for each other is
not very surprising.
What came up as a surprise was the
level of the affinity. When we tested in our affinity assay, the binding of the
sporadic CJD prions to the lipoproteins coated on the late, the mid-points
which was the indication of the affinity constants, they were in the low
picomolar range, between 30 to 100 picomoles.
That was a real surprise. The second
surprise was the selectivity. When we coated the plate with HDL, there was
practically no specific binding, no cooperative binding.
So, despite a similarity in the lipid
content between LDL and HDL, there was a big difference in the affinity for the
LDL, a preferential binding for the LDL.
The second surprise came from electron
microscopy when we purified the sCJD protein from the brain and then incubated
them with the VLDL and LDL, or HDL.
We saw decoration of the human neurons
only with LDL. We didn't see any decoration with HDL. If you compare the signal
of our best monoclonal antibodies, and decoration with LDL gold, we see how few
dots we actually got on those prion neurons.
On the other hand, we have a massive accumulation of the LDL on the
human prions.
The common component to all the VLDL,
LDL, IDL, and not HDL, is the apoprotein B. The other apo-C, E and so on, are
exchangeable, but they are not present in LDL.
So, we decided to test specifically
the apoprotein B, purify for protein B. The affinity was only about four to
10-fold lower than the affinity of the original LDL.
So, in conclusion, the PRP SC protein,
both the S and R forms, have a very high affinity for lipoproteins containing
apoprotein B, or apoprotein B itself.
The binding is conformationally
specific. If you compare the affinity constants of the alpha helical PRP versus
random cold PRP, versus native PRP SC protein in prion neurons, hey go in that
sequence.
So, alpha helical PRP doesn't have
practically any binding, random coil higher, and then followed by the native
prions.
The stoichiometry is also is also
different between recombinant PRP and the native prions, where we see the
binding ratio about three, we see only one to one ratio for recombinant PRP in
the random coil conformation.
The LDL suffers from a misconception. Most
of the people have the impression that LDL is cholesterol. That is actually not
true.
It is about 30 percent protein, which
is called apoprotein B. It has a molecular weight about 550 kilodaltons. It has
4,536 amino acids.
It composes about 30 percent of the
weight of the LDL particle. The rest is cholesterol and phospholipids in the
monolayer, and the apoprotein B, which is hydrophobic alpha beta sheets and
alpha helices, are basically wrapped around the particle and presents no
specific fusion with the cell.
If there wouldn't be apoprotein B, we
would die from atherosclerosis of age two or three, probably. So, it is a very
important mechanism which, through the LDL receptor domain, directs the LDL
particles to the cells, which express the LDL receptor and, if they meet,
influence of the cholesterol. So, it is
a very important regulatory mechanism.
Is it conformation specific also from
the other side? In the prions, when we
test the different prions from CJD, sporadic CJD, CN hamster(?), scrapie, RML,
we saw a totally different binding curve, indicating different stoichiometries
and different affinity constants.
So, it is not only conformation
specific for PRP, for human PRP, but it is also able somehow to discriminate
between PRP, different prion strains.
So, human LDL and apo-B binding with
AP CJD prions, it is conformation specific. It has a very low affinity constant
down to 30 picomolars.
The PRP affinity for binding is
present in 19 to 31. The order of the binding goes from the beta HPRP to the
denatured PRP to the alpha helical PRP.
The different stoichiometries, three
to one for native prions, one to one for recombinant PRP. The lipids of LDL are
not essential for the binding. Glycolipids and glycosylation are not essential
for the binding in PRP. LDL and APO-B
binding to denatured PRP is sequence specific.
So, did we look into sporadic CJD
cases. I think that the first step before that, we actually realized that first
we have to validate our assay.
We have to show that we have a -- that
we can truly detect PRPSC protein and, second, that we can truly
measure quantitatively PRPSC protein, and correlate it with the
prion infected.
So, in this study, which was actually
initiated with Glenn Telling, and whose transgenics he generated in San
Francisco, we inoculated three different cases of sporadic CJD in the end point
titration experiment in different transgenics to determine end point titers.
At the same time we made homogenase
from the brain and tested by CDI, the dilution curve, in parallel. When you see
the correlation, there is a very clear overlap.
It shows one important difference. The
50 percent transmission rate indicating one infectious unit per ml, at that
level, CDI has a reading skill of about 20,000, which is about 1,000-fold over
the capability of the CDI assay. So, in
effect, the CDI is more sensitive than the bioassay in transgenics.
So, one more question was correlating
the established procedures of immunohistochemistry and pathology with the
infectivity and with the CDI.
So, we blindly tested PRP C protein in
those different forms of prion diseases and in 18 different anatomical areas in
eight sporadic CJD cases.
We could detect the RPRPSC
protein everywhere. In contrast, the immunohistochemistry and localization profiles
in many areas the sensitivity of both was not exceeding 20 percent, or was even
lower than that.
So, one conclusion. First, the testing
has to be in a diagnostic aspect. It has to be in different anatomical areas.
Second, the CJCDI shows absolute diagnostic
sensitivity and specificity in all of those anatomical areas.
The second important finding was
related to the SPRPSE. When we looked at the concentrations of RPRPSC
versus SPRPSE, in all frontal and white matter areas we tested, there was more
SPRPSE protein over the RPRPSC protein. The RPRPSC protein actually formed
about five to 10 percent of the total.
The next presentation is going to be
humoral. This is just the first data showing the CDI on the VLDL fraction and
plasma of the CJD cases.
When we tested total PRP
concentrations in 21 donors and 20 sporadic CJD cases, we didn't find any
difference in total PRP or in the SPRP and the PRPSC protein. It is below the threshold.
When we separated VLDL, there was a
significant difference in the sense of more total PRP protein in the VLDL from
sporadic CJD cases, and most of the total PRP increase was RPRPSC
protein, actually.
So, what are the conclusions? I think the apoB containing lipoproteins are
strong candidates for carriers of sporadic CJD prions in human plasma, and I
will talk about it as the diagnostic implications emerge.
Binding of apoB containing
lipoproteins to native sporadic CJD prions is conformationally specific with
Kilodaltons down to 30 picomolars.
The existence of highly stable
lipoproteins prion complex in plasma suggests that it may be that the
lipoproteins have a role in the clearance of prions from the brain and other
tissues.
Both conformational specificity and
high affinity will lead to the development of new assays for prions.
The data on low affinity prion ligands
suggest that lipoprotein binding may impact the infectivity of sporadic CJD
prions.
Conformational specificity of the apoB
binding may lead to the new ways of differentiating human prion strains,
including variant CJD.
Plasma lipoproteins provide, in my
opinion, highly specific ligands for prion removal from plasma. Thank you.
DR. TELLING: Thanks, Dr. Safar. We
have time for maybe two quick questions before the break, if there are any?
DR. MASTRIANNI: I have got so many,
but I can talk to you later. One just
generalized question. Why does LDL bind scrapie better than PRPC? Do you have a conformational model for
that? Maybe I missed it.
DR. SAFAR: We don't have a really good conformation
model of apoB. ApoB is almost as
difficult to study structurally as PRPSC. It is very large, very
hydrophobic, and there are no three-D structures.
The model I showed is a computerized
approximation of the CD infrared spectroscopy of the entire EM. So, it is a
very large protein.
On the other hand, there are many
tools in molecular biology, including fragments of apoB and LDL, which will
allow us to determine which domains specifically, in the first approximation,
are responsible for the binding and we are studying it now.
DR. MANUELIDIS: If I understand it, you are saying that if
you take out the LDL fraction of plasma, you would lose a lot of the
infectivity in prions. Is that correct, and is that true?
DR. SAFAR: We think that practically all the PRPSC
from the CJD brains spiked into the plasma co-precipitated with LDL and VLDL,
yes. We haven't measured the
infectivity.
So, to your question, we measured PRPSC
protein, which we correlate with the CDI, which we correlate with viruses.
So, we are very sure that what we
detected is infectious PRPSC protein. Formally, we haven't done a
bioassay yet.
DR. TELLING: Thanks, Dr. Safar. I would like to keep this
on track, which we more or less are. I would like to take a break and reconvene
at 10:30.
[Brief recess.]
Agenda Item: Topic One:
Experimental Clearance of TSE in Plasma-Derived FVIII Products.
DR. TELLING: We are going to move on to topic one,
experimental clearances of transmissible spongiform encephalopathy infectivity
in plasma derived factor VIII products.
We are going to start out with
subtopic A, BSE clearance studies of factor VIII, study methods and clearance
levels, and a presentation by Dr. Scott from the FDA.
Agenda Item: TSE Clearance studies
for pdFVIII.
DR. SCOTT: Mine is the first of two presentations. The
second will be given by Dr. Thomas Kreil, representing the Plasma Protein
Therapeutics Association.
We are going to have somewhat
different presentations. What I would like to do is outline some of the
challenges in TSE clearance studies, and to introduce the committee to some
discussion questions that we would very much like your scientific input on.
Our TSE safety concerns are that,
theoretically, plasma derivatives might transmit variant CJD or other TSE
agents, since we certainly know that blood can do this and that plasma of
infected animals is also infectious.
Any such risk is probably very low, based
on the fact that no cases of variant CJD have been reported worldwide in any
recipients of plasma derivative, including in the United Kingdom where vCJD
risk is greatest.
However, we seek to assure the safety
of plasma derivatives, especially plasma derived factor VIII, against the risk
of transmission of TSEs.
The clearance of TSE agents in
manufacturing of plasma derived factor VIII and other plasma derivatives has a
major impact on estimated risk. In a
minute I will go through what I mean by clearance and clearance studies.
In a risk assessment, a draft risk
assessment that we published on the internet after the TSE advisory committee
meeting in 2005 for plasma derived factor VIII, that risk assessment had a
sensitivity analysis, which gives you an idea which inputs to the risk
assessment most affect the output or the level of risk.
Indeed, the clearance of TSE agents
during the manufacturing process is one of the major things that did impact the
ultimate estimated risk to recipients.
However, standardized methods for
studying TSE clearance in products have not been defined,in part because there
are a great number of challenges associated with standardizing these methods,
since we don't know everything there is to know about the TSE agents in blood.
We seek your advice about whether
standardized methods and assessment criteria are feasible now, and if they are
appropriate for determining TSE clearance in the manufacturing processes for
plasma derived factor VIII products.
In particular, these are your three
items for discussion, the feasibility and scientific value of adopting
standardized methods to assess TSE clearance in manufacturing of plasma derived
factor VIII products, and whether a minimum TSE agent reduction factor might
reasonably serve as an appropriate standard for demonstrating vCJD safety of
plasma derived factor VIII products.
If there is a minimum level that might
reasonably serve as such, what action should FDA consider if only lower levels
of clearance can be demonstrated for a given factor VIII product.
I am going to go into a little bit of
the previous history of TSE clearance studies and FDA's involvement in those.
Some of the members here today might
remember that we discussed TSE clearance studies with you in February of 2003.
Since then, we have engaged in case by
case review of the following types of information on TSE clearance. So , this
is information submitted by manufacturers officially to FDA, requesting a
labeling claim based on specific data that they had generated.
These studies include a rationale for
the animal model selected and a rationale for the spiking preparation. I will
get into some of these details in a minute, characterization of the spiking
agent, demonstration of accurately scaled down processes, robust and
reproducible experiments, well characterized assays for TSE infectivity.
These submissions will contain
estimated logs of TSE clearance by the processing steps that were studied, and
also will demonstrate or describe mass balance, that is, accounting for all the
input infectivity in the output samples that are assayed.
There should also be a demonstration
that mechanically similar clearance steps are or are not additive in the
process, and an accounting for conditioning of infectivity.
By conditioning, what I mean is, a
prior step in manufacturing might affect the physical state of the TSE agent
and, in turn, impact the amount of clearance that can be effected downstream in
the actual clearance step that is being studied. Again, I will go into this in more detail in
just a minute.
Since that time, four labeling claims
have been approved. These are for Carimune NF and panglobulin NF. These are
immune globulin products, and these are the reduction factors and these were
the steps that were studied, precipitations and nanofiltration for these two
reduction factors of 7.2 and 4.4.
Gamunex, another immune globulin
product is where a combination of cloth and depth filtration were studied and
the clearance level obtained was an average of 6.6. Thrombate III, precipitations were studied
with a reduction factor of 6.0.
Now, you notice there aren't any
plasma derived factor VIII products here. That doesn't signify whether we are
evaluating submissions or not, but it does tell you that no such studies have
been approved for labeling claims.
Why are we particularly concerned
about plasma derived factor VIII? Well,
we are concerned, of course, about all plasma derivatives, but
cryoprecipitation is the first in manufacturing of plasma derived factor VIII.
There are many other steps that may
follow as there is increasing purification to make the product. Dr. Kreil will
be talking about those more.
This is just a schematic of
Cohn-Onclay blood plasma fractionation process.
What you can see is the cryoprecipitate which becomes plasma derived
factor VIII comes off very early in the scheme.
So, one of the reasons to be concerned
is that there is not much opportunity for clearance like there might be for
albumen, which undergoes a series of sequential precipitations to be purified,
and immune globulins where the case is similar.
Many people have looked at
experimental clearances, either PRPTSE or infectivity by cryoprecipitation, and this
is just some of the references that have reported this.
You can see here that the log 10
reduction factor is really one log or less in all of these studies, and whether
a bioassay or a binding assay was used of a surrogate marker for infectivity.
So, these are the major challenges
that we face in standardizing these studies or even understanding how best to
do these studies.
I am going to talk about four things:
the exogenous or spiking experiments, endogenous experiments and their
relevance and feasibility, the TSE strain and animal model that is used, and
output measures of infectivity reduction -- bioassays, which are infectivity
assays in animals, and in vitro assays such as the western blot and the
conformation dependent immunoassay, which depends upon antibody binding.
This is a very simple schematic of how
an exogenous TSE clearance study might be done. The example I have given you
here is the cryoprecipitation steps.
So, here you would have plasma. You
would spike a preparation typically from brain of an infected animal into this
plasma, and then that would undergo the manufacturing step where you get
cryoprecipitate and cryopoor plasma supernatant.
The infectivity would be measured here
and here and compared to the amounts that you put in at the beginning to
determine a reduction factor.
So, for right now, I am going to be
talking more about spiking experiments. There are a number of studies that have
been done this way.
The reason that spiking experiments
are done with brain homogenate is that it tends to have a very high infectivity
level. So, you can achieve or
demonstrate a wide range of clearance values. It is practical.
The form of the infectious agent, a
number of these have been used, including membrane associated forms, brain
homogenate that is just centrifuged to clarify, ultra centrifuged preparations
including microsomal preparations and coevally like domains.
People have also used detergent
solubilized hemogenates from brain. There are also membrane free infectious
materials that have been used.
Again, they are derived from brain.
These would be fibrole preparations, but also more purified preparations of PRPTSE
.
In general, they can be somewhat
insoluble and they are felt, perhaps, not to be the best representatives of
blood infectivity, which is believed to be more soluble.
Here I am just going to show you some
examples of what we know about the spiking form of the agent. This is by Bey et
al, published in Biologicals.
This is just to demonstrate that the
form of the spike impacts clearance by precipitation. What you see here is the
manufacturing process, cryoprecipitation, two different alcohol precipitations,
and glycine precipitation.
These are the logs of PRPTSE reduction that were measured with respect to
the supernatant. This is microsomal spikes. You can see that, for
cryoprecipitation and glycine precipitation you have a fairly low clearance.
If you look at a more purified PRP
scrapie spike preparation, you get somewhat higher levels of clearance, here
two, three and four.
So, spike has a major impact on the
amount of clearance that you measure, which means it is very important to
choose, if you will, not necessarily the worst case spike, but perhaps the one
that might be most representative of infectivity in blood.
There is also the impact of
conditioning. This is an example of conditioning, where detergent treatment of
the infectious preparations diminishes its clearance by nanofiltration. This
was published in 2001 by Tateishi.
The feed solution is a starting
solution. So, this brain homogenate was treated with detergent or without
detergent, and these are the titers of infectivity that they got.
By the way, these experiments were
done by bioassay. So, all of these output measures are amount of infectivity by
bioassay.
So, this is what you begin with, the
detergent and non-detergent treated material. That was put through a 35
nanometer filter.
What you can see here is that
material, brain homogenate, that was not treated with detergent had a very nice
clearance by nanofiltration, 35 nanometers.
When detergent was added -- I think
this is sarcofil three percent, you get a much lower level of clearance.
So, what does that mean? That means that if you have a detergent step,
that that might impact the filterability of the agent at the end.
What you can also see is that, at
lower pore size filters -- that is, 15 and 10 nanometers -- you do get good
filtration in either case.
What this means for TSE clearance
studies is that you really need to consider the upstream steps that might
impact the agent.
You could get, in a sense, a result
that might not really reflect, if you only looked at nanofiltration and not the
upstream steps before, he impact on the agent, it means that you might
over-estimate the amount of clearance, for example, by nanofiltration.
This is one of the complexities of
doing these kinds of studies and one of the challenges faced by industry.
Another example of conditioning is PRPTSE clearance by membrane filtration and depth
filtration. This was shown in a paper by Van Holten, to increase in the
presence of alcohol.
It appears, from his data, that the
alcohol the used and the concentration of alcohol caused aggregation of PRPTSE
, which obviously influenced how well it was filtered by fairly large pore
sized membranes and depth filtration. It was clearly filtered much better in
the presence of alcohol.
I am going to briefly review the endogenous
infection model. In these models, plasma is taken from a TSE infected animal.
This is very low titer material,
somewhere on the order of a couple of IDs per ml to maybe 50 or 60, depending
on the animal model used.
It undergoes the manufacturing step
again, and you measure the amount of infectivity in the supernatant in this
case, or the precipitate.
The endogenous TSE clearance studies
have relevance to blood infectivity. I would point out, though, that the
comparison of results from endogenous and exogenous infectivity studies suggest
similar reductions for some precipitations, but the number of comparative
studies is extremely limited.
However, endogenous infectivity is
probably the most relevant to infectivity that we would find in human plasma.
The characteristics of endogenous
infectivity are thought to be a fairly small size of the infectious particle,
difficulty in sedimenting in its native form -- and by sedimenting I mean by
high speed centrifugation. It is
probably poorly aggregated and it may be lipid or plasma protein associated.
The relevance to human blood is highly
likely, but you can only demonstrate limited clearance because the starting
infectivity is so low.
That means that a large number of
donor and assay animals have to be used to compensate for these low titers. So,
in other words, if you have two infectious doses per ml, but you assay 100 mls
of material, you will likely find this infectivity.
Just to give you an idea, the volume
injectable intracerebrally to titrate this is only about 20 microliters --
sorry, .02 mls at 20 microliters per ml, or about 50 microliters per
hamster. For 100 mls of plasma, to
completely titrate it, you need 5,000 mice or 2,000 hamsters.
What is done in real life, I don't
know if anybody has ever done quite this many, but they will do a portion of
their output sample and look at infectivity and calculate essentially how much
they could have missed if they get a zero.
Large animal models, in theory, might
be nice to study. We know that sheep can have natural infection with scrapie
and can be experimentally infected with BSE.
We further know that the blood of
these infectious animals is infectious to recipient sheep. There are
experimental logistical hurdles in doing these kinds of studies
. Among those are herd management, and
the fact that there would be very limited locations where you would be allowed
to have a scrapie herd or a sheep herd infected with BSE. Furthermore, these would have to be very carefully
segregated away from the control animals.
These sheep also have very long
incubation times. So, you would have to wait even more years than you would
have to wait for a hamster or mouse study and they will be limited in
availability.
Of course, the logistics of scale down
would be different because you would have much larger volumes to work with, but
you would still have to use a pilot laboratory to simulate the manufacturing
process. Probably new or different pilot
laboratories would have to be set up to study clearance in large animals.
In terms of TSE model selection, there
are many papers that show that TSEs differ in their resistance to inactivation
but, to date, clearance of TSEs in plasma products has only been demonstrated
by partitioning studies.
The reason for that is that the
inactivation methods that have been used for TSE infectivity are very harsh
methods and you would destroy the proteins that you are trying to isolate and
purify for people to use by any of these methods.
There are very few direct strain
comparisons in TSE clearance study plasma derivatives, but alcohol
precipitations were looked at by Stenlin(?) and the group at Telecris. They found similar clearance levels using
western blot for BSE, CJD and vCJD spiked samples.
Alcohol precipitations, at least in
theory, could be influenced if strain related differences exist in aggregation
properties of the infectious agents.
This is a theoretical concern, but it
might also be a real concern, and we don't have the data to know whether or not
that is the case in these particular scenarios.
So far, strain differences for
partitioning clearance experiments have not been demonstrated. You can see how
limited in number the studies are.
What kind of assays should be used for
TSE agents in clearance studies?
Bioassay is usually done by limiting dilution titration into susceptible
rodents and, as you have already heard today, PRPTSE is felt by many to be a good surrogate marker
for infectivity, and this is usually measured by western blot or conformation
dependent immunoassay, as you have heard.
There is a rationale for retaining
bioassay use, because although binding assays detect PRPTSE , they
are examples of infectivity without detectable PRPTSE .
I should qualify that by saying very
often this has been PRPres, that is, PRPTSE as assayed by its resistance to proteinase K.
There are also examples of PRPTSE occurrence without infectivity and also I
would note that conditioning that I have shown you before might differentially
affect binding versus infectivity.
There is a paper by Silvera and his
group suggesting that, at least from brain homogenase, a certain size of prion
particle seems to be associated with greater infectivity, and that larger
aggregates and smaller aggregates are less associated.
So, even in the context of the protein
only hypothesis there are some caveats that one would have to consider in terms
of using PRPTSE binding
solely as a surrogate for infectivity.
Furthermore, binding assays currently
are not as sensitive as bioassays. We have just heard, however, that for the
conformation dependent immunoassay, this may be otherwise, and we look forward
to additional data in that respect.
The limit of detection for binding
assay, more typically, is two to three logs of infectivity. So, you can't
demonstrate as wide a range of occurrence when you are using, in general, these
kinds of assay.
An additional challenge in TSE
clearance studies is their interpretation, how much clearance is significance.
Well, we are going to ask the committee to discuss that, but I can give you
something to work with.
In viral validation or viral clearance
studies that are done for all plasma derived products in the United States, it
is typically demonstrated for effective viral clearance that there are at least
two to three logs greater clearance than the maximum potential absolute amount
of virus present. When I say the amount
of virus present I mean the amount of virus that would be expected in infected
plasma.
This added margin of safety is
probably important, because we don't know in every case how much virus might be
in infected plasma. We have a very good
ballpark estimate based on what is reported.
Furthermore, manufacturing itself has
its -- is not entirely robust. You might not get exactly the same value each
time you do a manufacturing step because of slight changes in parameter.
Again, this margin of safety, this
addition of more logs on top of what you think absolutely has to be removed, is
probably a good idea.
Now we come to TSE clearance. If TSE
infectivity is present in a unit of plasma, how much might there be. Well, if
you take an 800 ml plasma unit -- this would be the top amount you might
expect, and multiply that by the potential infectivity in it -- and we really
don't know exactly if these numbers are right -- so, these are estimates based
on other studies in the literature from animals.
So, you multiply by this range, two to
30. What you get is 1,600 to 24,000 range of infectious doses possibly expected
in this infected plasma.
That works out to 3.2 to 4.4 log 10
total infectious units. Actual infectivity might be less than this due to the
blood brain barrier and due to host susceptibility, but this gives you some
numbers to start with and to think about.
I am going to introduce the questions,
but Dr. Kreil will be following up with additional thoughts about TSE clearance
studies and more detailed information about where industry has been studying
clearance in plasma derived factor VIII.
We are asking you to comment on the
feasibility and scientific value of adopting standardized exogenous or spiking
study methods to assess TSE clearance in manufacturing of plasma derived factor
VIII.
We would like you to comment on your
thoughts on optimal spiking material and its preparation from the standpoint of
relevance to blood infectivity, the selection of TSE strain and animal models.
TSE immunoassays for PRPTSE versus bioassays for infectivity, the use of
these as output measures, and identification of manufacturing processes that
might alter TSE agent properties.
We would also like for you to comment
on the feasibility and scientific value of adopting standardized endogenous
study methods to assess TSE clearance in plasma derived factor VIII.
We would also like you to discuss
whether a minimum TSE agent reduction factor demonstrated using an exogenous
spiking model in scaled down manufacturing experiments, like the ones I have
described, might reasonably serve as an appropriate standard for demonstrating
TSE safety of the products.
Considering the outcomes to that
discussion in question two, in cases where only lower levels of clearance can
be demonstrated for plasma derived factor VIII products, what should we consider:
Labeling that would differentiate the
lower clearance products from other products with sufficient TSE clearance;
Recommending addition of TSE clearance
steps to the manufacturing method; Performance of TTSE clearance experiments
using endogenous infectivity models, or any other actions.
I will leave that with you and give
the podium over to Dr. Kreil. Thank you very much.
DR. TELLING: Are there any questions for clarification for
Dr. Scott at this time?
MR. BIAS: Dr. Scott, is there a reason that there
haven't been any experiments done using human blood of vCJD victims?
DR. SCOTT: That is a very good question. I think that if
this was easily available, they definitely would have been done.
I think by the time the patients come
to their clinical disease, the ability and the ethical constraints on
collecting a lot of blood or plasma from them has been limited.
In the United Kingdom, they are
particularly careful to assure that patients have a choice and that their
families have a choice.
That is what has caused the
limitation. It is not obviously the patient's fault. There aren't very many
patients to begin with, but there aren't very many people with this disease at
any given time that are in a situation where they might be able to give a large
amount of blood or plasma.
i know Dr. Minor is in the audience
and I wouldn't want to just call on anybody in the audience at random, but
either Dr. Asher or Dr. Minor might have more insight on the availability or
the potential availability of variant CJD blood. I guess I will let Dr. Asher,
because he is on duty here.
DR. ASHER: I hope that we will hear some thoughts on the
issue tomorrow from Dr. Minor who is here, and Dr. Turner who we expect to
arrive this evening.
The problem has been this. With
sporadic and familial CJD, infectivity has not convincingly been demonstrated
in the blood.
So, if you collected it, either by
epidemiological look back studies -- and the American Red Cross' study is
really now quite extensive -- and a very limited number of studies done at the
NIH transfusing whole blood into chimpanzees, none of whom ever became ill with
Creutzfeldt Jakob disease.
So, the blood from the forms of
Creutzfeldt Jakob disease generally available in the United States, the
hypothesis that there is enough infectivity present to be detected at all with
any of these assays has not been demonstrated.
With variant Creutzfeldt Jakob
disease, as Dot pointed out, the number of patients available has been very
small.
In the two cases in the United States,
I believe that Dr. Gambetti has a small amount of blood, and I know the
Canadian case there is a small amount of plasma available, but nothing
approaching what would be needed for the kinds of studies that we have been
talking about.
I am afraid at the moment we are stuck
with blood from endogenous infectivity. We are stuck with blood from animal
sources.
DR. GESCHWIND: So, at UCSF we have actually shown that it is
pretty feasible to get large volumes of blood from patients with CJD.
We have -- Jiri Safar probably can
give you the fact numbers, but probably we have over 50 patients in whom we
have gotten 200 to 400 mls of blood.
So, bring in patients from around the
country and at certain points when we have funding we have been sending out a nurse
to get 200 mls of blood from patients with CJD, and we have been collecting it
every two to three months from patients during the course of their disease,
depending upon -- we do very strict safety tests that are more conservative
than for the Red Cross blood donations, prior to doing this.
So, it is feasible, particularly in
patients whom we have diagnosed earlier in the disease course, and in patients
who have a slower course.
DR. SCOTT: I think that Dr. Minor also has a comment
maybe about the variant CJD cases.
DR. MINOR: Well, I am very jealous of the comment that
has just been made. I have discussed this extensively with the people at the
CJD surveillance unit in Edinburgh, and they won't touch it.
They basically say that the ethical
concerns are such that they will not take a unit from people who have variant
CJD, no matter who wants it.
I will be talking a little bit about
human samples tomorrow in the diagnostic presentation, and the availability of
human samples is absolutely tiny, relevant human samples, like within the
United Kingdom, is absolutely zero.
There has also been a recent
introduction of a thing called the human tissues act, which means that if you
don't do it right, you get sent to prison. That has actually been a major
inhibitory effect on actually trying to get these kinds of samples.
I am actually very impressed by the
fact that you can get those kinds of volumes around. If we could get those
kinds of volumes, I think I would put them into diagnostics rather than into
plasma fractionation, frankly.
DR. TELLING: What about blood from BSE
infected cattle?
DR. MINOR: This is like experimental infections you are
talking about or what?
DR. TELLING: Either experimentally infected -- well,
presumably that would be the most convenient source.
DR. MINOR: Again, I will talk about some of that stuff
tomorrow. There is a study which is going on with Ferna(?) Huston on sheep,
blood transfusion, where I think this is actually a kind of interesting animal
model for this.
The idea is that the sheep will be
infected by mouth by BSE or whatever, and then blood will be taken from them
and transfused into other sheep which are negative.
If you can actually keep a sample of
the blood which is transfused, and you can also follow the blood samples from
the transfused sheep -- I don't now how confusing I am making this sound -- you
can start talking about when the diagnostic tests become positive and when they
become negative.
You can also in theory, I guess, use
those kinds of materials for fractionating plasma proteins. I think the wrinkle
to that is it is not clear to me that plasma proteins fractionate from sheep
plasma in exactly the same way as they fractionate from human plasma.
So, there may be a doubt about even
the relevance of the model. I am sure
that can actually be done. I am not sure that has been done, but I think it can
be.
DR. TELLING: Any further questions?
DR. SAFAR: This is more a comment or offer. I think that
starting with Jim Mastrianni, who is at the table, and followed by Michael
Geschwind, it was a very difficult and challenging project, the logistics and
technical issues and the protocol issues.
With the help of NIH, with Michael
Nunn and many other people who cooperated, I think that all of those logistical
issues -- and that is an answer to Phil Minor more than anybody else -- it can
be overcome.
It took time and it was really
difficult, but I think that it is feasible to collect a significant amount, two
ml, 200 ml, at a session from CJD patients, either variant CJD or sporadic CJD.
So, I think that this is one of the
issues which should be discussed tomorrow in more detail, how to organize such
a collection and how such a repository should be handled, funded and organized.
DR. TELLING: With the obvious caveat that sporadic and
variant CJD may differ radically in their biological properties with respect to
infectivity in blood.
DR. SAFAR: Absolutely, yes.
DR. MANUELIDIS: I would like to make one comment about sort
of the definitive comment that David made about sporadic CJD.
It was shown in guinea pigs in 1978
that the blood is infectious and the spleen is infectious. It only makes sense,
really, that it would go to spleen if the sporadic CJD, the agent itself, went
through blood.
The second thing is that there were
two studies that were published in Lancet, one by our group and one by
Tateishi's group, showing that human blood actually transmitted as well.
Now, the Japanese group might have a
slightly different variant of their CJD because of the different geographic
region.
I think that probably the amount of
infectivity is much lower than it is in vCJD, but it is likely to be there,
from everything we know about these infections and the fact that spleen is
infectious.
DR. ASHER: We agree that in blood of patients with
sporadic CJD, infectivity is likely to be there, but the amount would certainly
have to be smaller.
Over 100 patients who received blood transfusions
from donors subsequently confirmed as having CJD followed for more than five
years by the American Red Cross, none of them came down with CJD, whereas a
very small number of recipients of blood components in the United Kingdom -- 18
-- got presented, three have already come down. So, it is clearly a very
different situation.
Now, I suggested for variant CJD that
cadaver blood for some purposes would be satisfactory, but the UK authorities,
apparently found that idea distasteful. You can get more than a liter of blood
from a cadaver, and privacy rights end at death.
DR. TELLING: Thank you. I think we had better move on to
the second presentation. Dr. Kreil, industry TSE clearance studies for factor
VIII.
Agenda Item: Industry TSE Clearance Studies.
DR. KREIL: Good morning, ladies and gentlemen. On behalf
of the pathogen safety steering committee of the Plasma Products Therapeutics
Association -- that is the group that I am going to talk on behalf of today --
I would like to thank you for giving us the opportunity to comment on some of
the aspects that Dr. Scott has raised in her presentation just a little while
ago.
This is just to remind you basically
of our constituency, worldwide manufacturers of plasma derivatives.
Specifically the considerations here focus on one class of products. Those are
the plasma derived factor VIII products.
The manufacturer, just to give you a
schematic of that, actually commences very early onwards from the plasma. This
is thawed up to just a little above freezing temperature, at which point, in
plasma, there is a precipitate that forms, the so-called cryoprecipitate.
By centrifuging these, precipitates
can be removed from plasma and that way you basically split up plasma into two
fractions, one being the cryosupernatant here, for the production of certain
coagulation factors, but then for also the classical Cohn products, Igs and
albumen.
Then the cryoprecipitate, where factor
VIII products are manufactured, historically they have been turned into high
purity. That is meant to say that, beyond factor VIII, they may contain albumen
factor in addition.
This is the principle of how TSE or,
for that matter, all virus or prion clearance studies are being performed.
So, you have a very large scale
manufacturing process. Typically we are talking thousands of liters. Obviously,
you cannot work with pathogens at that level for GNP considerations to start
with.
So, what is done is, we are scaling
down these processes into a scale that we can work with these processes in what
we call pathogen safe laboratories.
There we can work with biosafety level
agents, and what we do basically is, we run these processes at a very small
scale, typically upstream with a little less pure intermediate, a little larger
in volume, and then running through one of these purification steps.
What you get is typically a smaller
volume intermediate of higher purity. Now at the laboratory scale, what we can
do is, we can add upstream, for the purposes of today's discussion, prions but,
again, we are doing the same thing with all sorts of viruses also.
Then, after this is added, you go
through this manufacturing process at the laboratory scale. Then you can
determine the input of prion activity or prion surrogate markers and the
output.
Then, by comparing that, and taking
into consideration the volumes before and after, you can actually derive what
is the so-called reduction factor, so you can understand what reduction of
prions for that purpose is achieved by that manufacturing process.
A very important point obviously is,
this needs to be exactly like this. Otherwise, the numbers that we obtain are
not meaningful for the large scale production of biological medicinal products.
That is why a lot of time is spent,
once you have established the so-called downscale, into validating that down
scale.
Really what we mean by validating that
down scale is that we are going to validate that this downscale is equivalent
to the large scale manufacturing process, because this, again, is the
fundamental principle under which we can derive knowledge about the large scale
processes, from doing these small scale experiments.
So, what can be done to make sure that
the information that we derive at the small scale is meaningful? Well, first, the intermediate that we use for
running these small scales is directly derived from our manufacturing
facilities, or is somewhat specialized materials and we get them maybe from a
pilot scale.
In other words, this is regular
intermediate that would be manufactured into commercial product. Now, for this
product, a number of different parameters can be assessed, not only for the
input, which is equivalent anyway by means of its origins, but also, after
doing the small scale purification, you can determine whether the amount of
protein concentration or activities, for the purification that supposedly
should occur in the large scale, does also occur in your small scale.
Further, a number of process parameters
can be monitored and, as you can see from the numerous examples on this slide,
these do vary depending on the step that we investigate.
If it is a precipitation step, then
obviously the concentration of the precipitating agent, the time that the
intermediate is stirred with this agent, the temperature would be important.
Then, for example, calling for other
steps, then things like pH, conductivity, ionic strength or contact time with
the resident would be more important.
Again, this is sort of the
prerequisite under which we have to operate. So long as you can't demonstrate
that there is a perfect equivalent of the small scale with the larger scale,
all further information that you derive would be meaningless for the large
scale.
Now, for prion clearance studies
specifically Dr. Scott has pointed out already that there are a number of
choices that one needs to make before going into a prion study.
There is first the choice of a spiking
agent from which organism we want to derive that. Secondly there is a number of
different possibilities for particular spikes for the initial spike
preparation.
Initially people have used the organ
that does contain typically the highest levels of prion levels -- the brain --
and have more or less purified that as a spiking material.
So, brain homogenous has certainly
been the first material used, and then different purification forms off that,
such as microsomal fractions, detergents treated or sonicated intermediates of
that.
Finally, there is a choice of which
assay you want to use for a readout of your prion clearance study. That would
be either in vivo, which is cost intensive, resource intensive, and you will
have to wait a long time to get a result or, alternatively, in the in vitro
assay such as the western blot or the CDI.
Now, it is important to understand
that, for these prion quantifications, there are a lot of controls also put
around this such as, for example, physical reactions are quality control.
There are good laboratory practices
applied and, where it is not possible to become certified for the application
of good laboratory practices for such studies, and that, as I said, is not
possible in all geography. Then at least the principles are being followed.
Then finally, the preparation of the spike
materials and how the assays are performed, when an assay is acceptable or not.
All of this is writ down in what we
call standard operating procedures so that there is a lot of control being put
around the reproducibility of these assays.
Obviously also, such as with every
good assay, controls are being put in place, such as a positive control,
negative controls, controls for interference of the matrix with the performance
of the assay, et cetera.
So, I guess we can say that, by
putting in place all of these controls, we can certainly guarantee that the
assay is suitable for the purposes of a prion reduction study.
The agency has asked the question
whether further standardization or a validation of such assays would be useful,
and we would like to make a number of arguments why we believe that that would
be useful.
First, I have to mention that, during
these manufacturing processes, it can be observed that the initial spike is
being conditioned through the process, such that actually you would want to use
different materials for investigating different manufacturing processes.
If, for example, you had a solvent
detergent treatment upstream from the process that you want to investigate,
then any prion that would have been present in plasma, that would have come
down to the step that you investigated, would have gone through that contact
with solvent detergent.
So, it might be a chance to consider
using a spike that has been detergent treated. That would reflect, probably
most adequately, whatever was upstream in the process.
Also, investigating the potentially
additive effect of sequential steps will require you, for example, to do a run
without subsequent spiking. So, again, standardizing how exactly you need to do
that experiment is going to make the interpretation difficult and will limit
your ability to demonstrate removal.
That is why we would argue that it is
more important to rely on good expert judgement first, and then obviously also
justification of that judgement on a case by case basis.
Another useful example, we believe, to
look at this -- and again, Dr. Scott has shared with you the very same example
that I will bring up here -- it has been shown that there can be very
substantial removal here by log steps, for example, using a filter with a
nominal core size of 35 nanometers.
In the presence of detergent during
the preparation of the prion spike, however, that removal becomes less
significant, and on the smaller core sized filters, we have been able to remove
the spike.
Now, further data that are available
and yet unpublished have shown that if you use more drastic detergent treatment
and sonication of the prion spike material, then in reality you can get the
material even through a 15 core sized filter, with virtually no removal at all.
So, one might argue that that would
then be a worst case and such a spiking material should be used for the
evaluation of nanofilters.
So, our belief is that these
conditions are certainly very important in trying to understand the elementary
nature of the infectious unit for prions, but I think these should be seen as
experimental conditions.
During manufacturing, we do certainly
not add these high levels of detergents, and certainly we do not sonicate our
intermediate.
Therefore, should any prion agent be
present in plasma, then it would not be sonicated detergent treated to the
degree that has been used in this more experimental set up, to understand
better the nature of the agent.
That is why we believe that the
reduction capacity for nanofiltration has been widely demonstrated under more
relevant conditions for manufacturing.
There are some recent, I would like to
call it, advances in science that would suggest that maybe we should be using
different spiking materials to the ones we have used so far.
A very recent piece of evidence has
come from an Italian laboratory where it was demonstrated that starting from
brain homogenate at a titer of roughly 108 infectious units per ml,
after a very high spin, you can actually device material in the supernatant
here, and that supernatant still has a very high level of infectivity, yet very
little or even no PRPres demonstrated by, for example, western blot.
That paper, in the conclusion this
should suggested that there should be a suitable spiking material to use in
validation.
Now, some of the limitations there
already are, that you would only be able to do in vivo assays because if there
is no PRPres even in the starting material, then that would not be a good
readout for a reduction study.
Another complication, however, is that
this 105, while still a reasonable titer, represents only
one-thousandth of the original input.
So, it is a tiny little minority of the original PRP agent.
Now, that we have seen already in an
earlier study, where endogenously present infectivity has been fractionated
using a less drastic centrifugation here.
Even for endogenous or, if you will,
the relevant former sensitivity, you can see that with centrifugation you can
pellet quite a bit of that infectivity.
So, the question becomes, if you are
looking at these infectivities, are you interested in the majority of the
infectivity or do you want to investigate a tiny little minority that, in
behavior, may not at all reflect what would be present even in an endogenous
infectivity situation.
Another piece of recent information, a
very elegant paper has been published recently that I would like to discuss
because it seems to have pertinence to the conduct of reduction studies.
It would be an experimental model, a
transgenic mouse model, where these might express PRP without the GPI anchor.
Now, if these mice are infected with
prions, then they do not develop a classical pathology of scrapie. What is
interesting is that they have very high levels of infectivity circulating in
their blood.
One might argue that that would be a
high titered blood spike so something rather usable for validation
studies. Certainly this has been
suggested to be the case by the authors.
I guess one argument that I would like
to convey to you is that this PRP protein is devoid of the GPI anchor and,
therefore, this truncated version is of unclear relevance to the
pathophysiologically relevant prion agent that w are concerned with, should it
really be demonstrated to occur in plasma.
I would argue, if we investigated the
removal of this truncated form, then those results might, because of the
similar or dissimilar nature of the agent, tell us something about the true
agent or might not tell us something.
In summary, we feel that certainly
through the validation of equivalence between large scale and down scale, the
controls we put around all the prion spiked materials and also the controls
that we put around these prion assays, prion clearance studies as we have
performed them up until now certainly have generated meaningful information.
We feel that, therefore, further
standardization would, in fact, inhibit process specific investigations more
than anything else.
We feel that we should more rely on
expert input, obviously providing the adequate justifications. Further, given the enormous advances that
science comes up with at a very rapid pace -- the two papers that I have just
shared with you have actually been published in the last two months only --
would also prevent using novel approaches that might allow us to investigate
more meaningful processes, and I think would really discourage, more than
anything else novel approaches.
As Dr. Scott has mentioned, we are now
going to share with you a summary of different prion clearance studies that
have been performed throughout the industry on specifically plasma derived
factor VIII products.
Before showing that to you, I would
like to make a number of qualifiers. You
need to keep in mind that not all of these products are manufactured using the
same manufacturing process.
This also results in different
clinical usability, such as some of these products contain non-relevant factors
in addition to factor VIII and therefore cannot be seen as typical or just
another factor VIII product, if you will.
Also, for the reduction factors that
you are going to see for the overall clearing, it is not necessarily so that
all the manufacturing steps have been investigated.
So, a lower clearance factor may just
mean that not all of the steps have been investigated. Should that be done, the
numbers could be much different.
Also, we would like to point out that,
for the products that have been licensed in the United States -- and that is
mentioned in the footnote of the slides -- these data have been shared in more
comprehensive fashion with the agency.
We would also like to point out that,
at this point, there are a number of research studies going on. The results we
will await and will provide further results.
So, this is the first one. We have
taken a look at two manufacturing processes that have been investigated, one
being purification with a monoclonal antibody column, and then there is another
ion exchange chromatography column.
This is since the 263K strain of
scrapie adapted to hamsters have been used, with an infectivity assay. So,
bioassay was used for generating the numbers here. You can see a total log
reduction of roughly eight logs was demonstrated.
Here is another product, also licensed
in the Untied States. Here four different manufacturing processes have been
investigated -- actually three, I apologize.
That is a PG precipitation here, another affinity chromatography step
here, and then a final precipitation plus final filtration.
What has been used here are two
different preparations of spike, one being a microsomal preparation here and a
detergent 3-D preparation here. Here, the same detergent 3D preparation with
the brain homogenate as a complement, and again here, the microsomal and the
detergent preparation.
Two independent runs have been
performed first by preparation and the mean reduction factors you can see down
here. The product overall has a demonstrated safety margin of greater than nine
logs of prion.
Another product here has investigated
two combinations of steps, one being a sequence of precipitation procedures,
and the other a sequence of chromatography events.
There have been, in this instance
here, two spike preparations used, here one, and two independent ones, first by
preparation, have resulted in these reduction factors here. It should be pointed out that this product is
not licensed in the United States.
Another product from the same company,
again not licensed in the United States, again, sequential procedures have been
investigated here.
Here the spike preparation is
mentioned here, in a single run, again using the western blot in vitro assay,
if you will, with a cumulative roughly six log reduction for these products.
Company D, that product is also
licensed in the United States. Again, two different spike preparations have
been used, a purified PRPSC or microsomes.
It has been assayed with the CDI assay
and two runs were performed per spike preparation, resulting in these mean log
reduction factors here.
A further product that is licensed in
the United States, here is the sequence of events, if you will, when one goal
has been investigated. So, sequential steps.
This has been done with two different
spike preparations, and one run per spike preparation was performed with a mean
reduction factor of 3.7 to 3.8.
Finally, this is the last product,
again not licensed in the United States. Here, again, another sequence of steps
has been investigated with a single spike material, brain homogenate, and that
resulted in a 3.5 log reduction.
So, summarizing all of these already
summarized data, I would like to point out that we feel that manufacturing
processes for plasma derived factor VIII products do remove prions, to varying
degrees.
The individual reduction factors that
we had on the summary slides really depend on, first, the specific
manufacturing process. That is also
resulting in different product quality, if you will. Secondly, obviously these
numbers depend on the number of steps that have been investigated. The more
steps investigated, the higher the numbers.
Finally, to some degree, on the
experimental design. Using in vivo assays, for example, allows you with a
higher dynamic range to demonstrate larger reduction factors. So, there may
just be larger reduction factors inherent to the assay system that you use.
In summary also we feel that, in terms
of the safety margins of these products, it is important to point out that the
level of risk at this point remains unknown, the specific level of risks, but
very likely the level of risk is low.
There is not any evidence for the
transmission of prion diseases by plasma derived factor VIII products, andthat
despite the very high level of pharmaco vigilance, I would like to mention the
multiplication exercise that the United Kingdom has gone through.
Patients, where it is known that their
product has been derived from also the contributions of latent bearing(?) CJD
donors have been notified of their presumably increased risk, and these people
are being closely monitored.
Epidemiologically, I think it is also
important to point out, as Dr. Scott has also mentioned, that the exposure is
low and the exposure seems to be getting lower still.
There is, as I did hopefully convince
you, a reduction of prion agents by all the plasma derived factor VIII
manufacturing processes that I have shown to you.
Therefore, we feel that the
quantification of reduction versus an unknown certainly low level of risk is an
open equation at this point, really.
In conclusion, we would like to say
that, given the unsubstantiated level of risk associated with plasma derived
factor VIII, we feel that this is not a rational basis for implementing further
measures, because it needs to be kept in mind that any additional steps that
might be implemented might also adversely impact the product characteristics,
starting with clinical safety, but then with additional manufacturing steps,
also typically yields suffer and, therefore, availability may be affected.
I can say on behalf of industry that
certainly we continue to be committed to research. We have done these studies
on a voluntary basis and, as I said, further studies are currently being
conducted and results will be made available. Thank you very much.
DR. TELLING: Thank you, Dr. Kreil, for that perspective.
Are there any questions at this point, or comments?
DR. HOGAN: Tom, I can assume, then, that the products,
after these additional steps, are all biologically active and have been
evaluated for safety?
DR. KREIL: The steps that I have summarized for you this
morning are the steps that are being conducted during the manufacture of
commercially licensed product.
So, all of these products are
clinically usable because they wouldn't have received a license otherwise. It
is just that history has shown that, whenever manufacturing processes are
changed, such that, for example, greater virus reduction is afforded, that
typically that results in a reduced yield of these product, and clinical
usefulness of the product then needs to be reestablished by clinical trials.
DR. HAMILTON: What concern is there about the exceedingly
long incubation period for CJF and variant CJD, and also could you speak about
leukoreduction effectiveness?
DR. KREIL: Well, regarding the long incubation period of
variant CJD, this is one of the aspects that still will not allow us to come up
with a final judgement, I guess. It is one of the uncertainties. We just need
to wait for further advancement and understanding of these diseases.
This is why we don't say that there is
categorically no risk, because I think at this point we cannot say this.
In leukoreduction, leukoreduction has
been very elegantly investigated for the reduction also, prion activity.
As far as published data,
leukoreduction has actually been shown to just result in a marginal reduction
of prion infectivity.
There is no further research going on
to enhance this leukoreduction to provide the filter with an added prion
removal capacity but, to my knowledge, these devices are not yet available.
DR. HAMILTON: Could you compare this rarity or lack of --
this low incidence in this situation to the low incidence that was supposed in
the early 1980s with HIV?
DR. KREIL: That is a very difficult comparison, I think.
Certainly with HIV, very quickly, during the early 1980s it was realized that
there was a blood transmissible agent there.
While the virus was not known at that
point, research did quickly establish its presence, and actually the virus was
present at very high levels, as we know.
To compare this with prion agents
where, despite intensive research, the demonstration of presence in plasma has
not been successful, I think would be very different.
I mean, certainly the levels of risk
are very different. It has been mentioned today that, out of 18 potential
opportunities for transfusion transmission of variant CJD, three have resulted
in a transmission, which is very different from viral diseases as we know them.
There you would be looking at a 100
percent transmission likelihood, if we took the figures of blood and transfused
it into a recipient. I think this
comparison would not be appropriate.
DR. BROOKMEYER: could you comment some on the reproducibility
of some of those reduction factors of the data that you showed?
Some of the data, it looked like there
was only one independent run or one or two independent runs. If you could just
comment on the variability and also on how much you think those reduction
factors depend upon the input and how much is actually being spiked in. What
are the sources of error or variability in those reduction factors?
DR. KREIL: I was trying to point out that obviously we
do everything possible to control these experiments well.
This is what scientists do. At the end
of the day we like to have information that is meaningful. We are not trying to
make up numbers, if you will.
Regrading the reproducibility, you are
right that some of these experiments have been informed with an N of one, but
others have been performed with numbers of repeats.
I can tell you that these repeats have
been very reproducible. So, I certainly do believe that the studies as I have
told you have been controlled adequately to ensure that the numbers are, first,
reflective of what occurs in large scale and, secondly, have been adequately
controlled so that the numbers are meaningful.
Now, all the limitations, all the
caveats that Dr. Scott pointed out are acknowledged. I mean, we haven't
seen the agents occur in plasma. Therefore, we don't know exactly what it would
look like in plasma, should it occur there.
So, all the spiking materials that we
are currently using are models. Therefore, the specific number with the agent,
would we have it, as it occurred in plasma, would it occur there, might look
slightly different.
I guess one important point to mention
is, all these numbers are numbers on a log scale. So, if a number is 3.1 or 3.3
quite frankly, for all practical purposes, the same thing.
In a log scale, again, there are
potencies of 10 that you are measuring against. So, minor differences that
might occur during experimental set ups would be insubstantial versus the
reduction factors that we have seen.
DR. TELLING: If there are no further questions, thank you,
Dr.Kreil. Next on the agenda is the open public hearing. Bill, would you let us
know who is registered for the open public hearing?
Agenda Item: Open Public Hearing.
DR. FREAS: As part of the advisory committee procedure,
we hold open public hearings so that members of the public can address the
committee on issues pending before the committee.
Mr. Chairman, at this time, I have
received four requests to speak in the open public hearing sessions, one
request for this morning, two for this afternoon's sessions, and one for
tomorrow.
I would like to invite the speaker for
this morning, Dave Cavenaugh, government relations for the Committee of Ten
Thousand, up to the podium.
While he is coming to the podium, Mr.
Chairman, I would like you to read the open public hearing statement required
for the meeting.
DR. TELLING: Both the Food and Drug Administration and the
public believe in a transparent process for information gathering and decision
making.
To assure such transparency at the
open public hearing session of the advisory committee meeting, FDA believes
that it is important to understand the context of an individual's presentation.
For this reason, FDA encourages you,
the open public hearing speaker, at the beginning of your written or oral
statement, to advise the committee of any financial relationship that you may
have with any company or any group that is likely to be impacted by the topic
of this meeting.
For example, the financial information
may include the company's or a group's payment of your travel, lodging or other
expenses in connection with your attendance at the meeting.
Likewise, FDA encourages you, at the
beginning of your statement, to advise the committee if you do not have such
financial relationships.
If you choose not to address this
issue or the financial relationships at the beginning of your statement, it
will not preclude you from speaking. So, Dr. Cavenaugh?
Agenda Item: Statement by Dave Cavenaugh.
DR. CAVENAUGH: My name is Dave Cavenaugh. I am government
relations staff for the Committee of Ten Thousand.
I think as many of you may have heard
in one place or another, our president, Cora Dubin, likes the expression coined
in the last year or so of having an arm in the game.
All during this discussion I have been
sitting here and thinking we have all these studies that show that prions can
be reduced in factor.
We don't have a lot of information
that they are being reduced in factor. That factor that is going out now, the
factor that is going into people's arms, is still the same factor that it has
been.
Today's discussion of prion reduction
through fractionation brings little comfort to persons with hemophilia in the
United Kingdom.
Different interpretations of the
findings of science show the United States and the United Kingdom to be going
down very different roads on this subject.
As long ago as 1999, when the FDA
first announced that screening for classical CJD was no longer needed, the
agency began distinctly identifying the greater perceived safety of plasma
products to whole blood:
"...experimental studies in
animal models for CJD suggested that manufacturing procedures used for plasma
derivatives could lower the amount of infectious material present in plasma
derivatives compared with whatever levels could be present in blood."
In 2002, FDA moved further down this
road:
"...we recommend that you defer
donors of whole blood and blood components intended for transfusion. source
leukocytes and recovered plasma, but not donors of source plasma, who have
resided in Europe for a cumulative period of five years or more, between 1980
and the present."
This exemption of plasma occurred when
the whole blood geographic donor ban was first being expanded beyond the United
Kingdom.
Since that time, there has been no
retraction from this position by the agency. Thousands of units have been
collected throughout Europe, pooled and fractionated to make factor VIII, IVIG,
albumen and other products, which of course by now have all been consumed,
largely by Americans.
It was in 2003 and 2004 that true
cases of vCJD transmission by blood were reported in the literature. From that
point on, all of the language of prior government and industry regarding
theoretical risk of transmission became obsolete.
The United Kingdom, following a
different time line regarding discovery of the dangers of variant CJD in blood,
learned of the contamination of plasma pools years earlier, and declared the
recipients of products from those pools to be at great risk.
The now famous 2004 letter from the
Ministry of Health to 4,000 homes of persons with hemophilia instructed them
not to donate blood, organs or tissue, and to inform medical, surgical and
dental providers, so that disposable instrument can be arranged for in advance
of any procedures.
Two years have passed since the risk
communication exercise in the United Kingdom.
The stigma it brought to every family with hemophilia is somewhat dulled
now, although it was unprecedented and disruptive for weeks at the time.
So, which is it? Are we at great risk
or is there no risk, or rather undetectable risk, which is not the same thing?
CBER advisory committees are often
asked to decide on issues for which there is inadequate data to make sound
judgement.
COTT has watched this country's
response to TSEs unfold, from the USDA's denial that there is any problem to
the untracked venison eaters in areas of US CWD outbreaks.
We ask that you do not give ground. Do
not expand the exemption from geographic donor bans which plasma collection now
enjoys. We further ask that you retract altogether this dangerous exemption of
source plasma from geographic donor bans. Thank you.
DR. TELLING: Thank you very much. Are
there any questions or comments? Is
there anyone else in the audience who would like to address the committee at
this time?
Okay, I thank everybody. We can
adjourn until 1:00 o'clock for some lunch.
[Whereupon, at 11:50 a.m., the meeting
was recessed, to reconvene at 1:00 p.m., that same day.
A F T E R N O O N S E S S I O N (1:05 p.m.)
Agenda Item: Open Committee Discussion.
DR. FREAS: Before we begin the afternoon session, Mr.
Cavenaugh has asked to address the committee for a brief announcement.
MR. CAVENAUGH: I would like to correct some possible
misinterpretation of what I said before, just the part about the dissimilar
geographic donor ban between whole blood and plasma in Europe creating a lot of
blood collected in Europe from countries that don't have the ban and brought
here from processing. That doesn't occur, and I know that.
People who live there for some time,
who are American, live over there six months or more, in many of the countries
to which we now have bans, come back here and give plasma, and that is
basically exposed to European risk factors.
The other is definitely correct.
DR. TELLING: Thank you. Dr. Epstein?
DR. EPSTEIN: Let me just state it in my own words. I agree
with what Dr. Cavenaugh just said. The committee needs to understand that
plasma for fractionation into U.S. licensed products has never been sourced
outside of the United States.
DR. TELLING: Are there any other comments? If not, thank you. So, the next item on the agenda is open
committee discussion. Dr. Scott, would you like to rephrase the questions for
us?
DR. SCOTT: Would you like me to go through all of them
or just one by one?
DR. TELLING: I think one by one for now.
DR. SCOTT: So, the first question is, we are asking you
to comment on the feasibility and scientific value of adopting standardized
exogenous or spiking experiment study methods, to assess TSE clearance in
manufacturing of plasma derived factor VIII.
We are asking you to consider and to
comment on what is the optimal spiking material and its preparation from the
standpoint of relevance to blood infectivity.
Second, whether you feel there is any
particular preference for TSE strain or animal model in these types of studies.
Whether immunoassays for PRP TSE --
well we would like for you to comment about the use of immunoassays for PRP TSE
in the context of these clearance studies, as compared with bioassays for
infectivity.
Lastly, identification of
manufacturing processes that might alter TSE agent properties. We would like
for you also to comment on that based on what you heard and perhaps other
things that you know about the agents.
DR. TELLING: Thank you. So, before we get an answer, let's
discuss the question as it stands. I would like to make it open to the
committee for discussion right now. Does
anybody have any comments or additional questions?
DR. SALMAN: This is a point for a clarification. I
thought we had discussed in the previous sessions what FDA has done with the
risk assessment.
To my knowledge, the risk assessment
led to the conclusion that there is almost like very low risk or negligible
risk related to the factor VIII. Maybe
somebody from FDA can clarify that.
DR. TELLING: Could somebody from FDA comment and clarify
on that?
DR. EPSTEIN: Thank you very much. Actually, in the October
2005 TSE advisory committee meeting, we discussed the model that FDA would
apply to estimating the vCJD risk from U.S. manufactured factor VIII.
We didn't actually show an output of
that model. We do intend to bring to a forthcoming meeting of the committee the
results of that assessment.
Generally speaking, looking at the
input parameters, we think the risk will be lower than for products made in the
United Kingdom, but we have not actually brought forward yet and output of the
risk assessment for U.S. licensed factor VIII.
DR. SALMAN: Thank you for the clarification. I think now
that you remind me, you are right. Would you think it is better to try to do
the risk assessment before we decide about the protocol for how you assess the
clearance of the plasma?
DR. EPSTEIN: I don't know that that is a question that
just one person should answer. We do think that the risk assessment is highly
informative.
The main message, though, is that the
factors that most affect the assessed risk are the clearance, the prevalence in
the donor pool, and the product usage in the patient community.
Far and away the largest variable
really is the clearance. That is why we felt we need an antecedent discussion
about just how well do we understand clearance and how concerned should we be
about the absence of standardization in the data that has been brought forward
to date.
I am not saying it is an absence of
quality work. As Dr. Kreil pointed out, there are a lot of efforts made to
assure high quality experimentation, but there is a lot of underlying
variation. That is what we are trying to get at today.
I understand your point, that these
things play back and forth against each other, and it is an open question.
DR. SALMAN: One issue here is important. The first step
in the risk assessment is hazard identification. As you stated in the
beginning, the plasma source sold in the United States or marketed in the
United States comes only from the United States.
To our knowledge, we don't have any
native new variant CJD cases in the United States. Is that correct or not?
DR. EPSTEIN: That is correct. There are two cases, both of
which are thought to have been acquired abroad.
However, as Mr. Cavenaugh correctly
stated, there are persons who have had exposure, residency or travel, in parts
of Europe where BSE has been prevalent and where vCJD has been reported.
Some of those persons may donate in
the United States. For example, a person may have resided in the United Kingdom
between 1980 and 1996 but for a period of less than three months. That person
is not deferred as a plasma donor.
For instance, a person might have
resided in France between 1980 and present but less than five years. That
person might not be deferred as a plasma donor.
There are persons in the United States
who have had some level of exposure. However, the likelihood of donation by
someone infected with vCJD as a U.S. plasma donor is certainly much lower than
it is in Europe.
DR. LEITMAN: Could I follow up on that question which I
think is very important, which I am having trouble grappling with?
In the United Kingdom hemophiliacs,
one would think they would be at the very highest risk of having acquired
variant CJD through the infusion of clotting concentrates, especially from 1980
to 1996.
Is it not correct to say that there
has never been a single reported case, including post-mortem studies of brain,
of subjects who died of other causes with hemophilia?
So, even in the best prospective
surveillance system in the world, which is probably the United Kingdom, that
has not been demonstrated; is that correct?
DR. TELLING: That was my understanding from this morning's
comments, yes.
DR. LEITMAN: That has to be part of the hazard analysis.
So, compared to that hazard in that population, I don't see how you could even
make an assessment of hazard so small in U.S. recipients of clotting factors.
I have trouble with all of those
questions because there are so many other more pressing issues to address than
the ones we are asked today.
DR. SCOTT: I would like to make some additional
comments. The first is that, although it is true, as far as we know, nobody who
has received a clotting factor or other plasma derived product has come down
with this disease in the United Kingdom.
I think it is useful to remember that
even in people who receive at least a whole unit of infected blood, it took
them six and a half to eight years to come down with a clinical infection.
Now, you would imagine that the amount
of material that they received through infected blood would be quite a bit
greater than what they received through a plasma derivative, which perchance,
would have some clearance during manufacturing.
Since these diseases have very
prolonged incubation times, I think we can't really say for sure whether or not
people have acquired this infection from clotting factors.
That is part of what drives our
concern. Also, I want to comment that, although we don't have the results of
the factor VIII risk assessment for you today, that what we are asking you
really for is a scientific opinion about how we might think about and address
these particular questions that are at hand, about how to do, if you will, the
best or the current optimal TSE clearance study.
We are not asking you to tell us
exactly how to standardize it. We are asking you for your opinion about what
you think, based on your scientific evaluation, might be useful for us to know
and consider in looking at this.
DR. TELLING: Dr. Epstein, and then maybe we should go
through the points in order.
DR. EPSTEIN: Dr. Scott has covered the main point that I
wanted to make, which is that the absence of cases doesn't rule out long
incubation periods in people who got exposed to a very low level of infectivity,
and that is the source of the continued concern.
DR. TELLING: So, these concerns notwithstanding -- we have
got one more question here.
DR. BROOKMEYER: I had a question about the risk assessment.
The impact of errors on the estimated clearance rate on the risk assessment, if
you are off by -- I am trying to get a sense of how accurate you need to get
that clearance rate.
If you are off by, say, a factor of
two on the clearance rate, not on a log scale, what would that do to the risk
assessment, to your estimate of risk?
I don't know if someone from the FDA
could comment on that in terms of is the error directly translated to the risk
assessment.
DR. TELLING: Dr. Scott might be able to. It may be that at
the next meeting, where we actually discuss the risk assessment model, it might
be the more appropriate venue to do that, but if you do have some comments
along those lines and if you would like to share them with us?
DR. SCOTT: I think we can't share numbers, but at the
last meeting we discussed with the committee how many estimated logs of
clearance or how many stratifications and ranges to have in the risk assessment
analysis.
So, the choice was two to three logs
clearance versus four to six versus seven to nine. You -- I expect you will be
able to see that, but certainly a two-fold difference wouldn't have a great
impact, but the sensitivity analysis, since it identified this as having major
impact overall, the logs difference of clearance may be important.
DR. JOHNSON: Glenn, maybe you can clarify this. One of the
issues that comes up, certainly in answering both number one and number four,
is the effect of aggregation on infectivity, and how much more aggregation
there is likely to be if it is prepared from brain, which is the only place
where you could demonstrate those kinds of log changes, and a lot of these
things are hinged. Do we have any good data on aggregation?
DR. TELLING: I think there was some evidence this morning
presented suggesting that you can sediment infectivity but still recover
significant amounts of infectivity, nonprotease resistant, in the supernatant.
I think that may be addressing somewhat your concern, but I think beyond that I
am not quite sure if there are any firm data on that issue.
DR. JOHNSON: But it really has to be -- you have to
bioassay that infectivity.
DR. TELLING: That is correct.
DR. JOHNSON: I think if you show the
western blot activity being one place, the other is not sufficient in my mind.
DR. TELLING: Weren't those data referring to infectivity
assays? I am sorry, I am talking about
the last statements that we heard, the last evidence, Dr. Kreil?
Can you discuss some evidence
suggesting that you could sediment material and recover significant amounts of
infectivity in the supernatant? I guess
it would be equivalent to SPRP scrapie; is that correct? Was that by bioassay?
DR. KREIL: That was by bioassay. In that supernatant, the detection by western
blot was attempted in two -- there were two attempts in detection by western
blot, one successful and one not successful.
So, there is very little PRPSC,
the proteinase resistant form, but there is substantial ambiguity in that
supernatant.
DR. TELLING: Thanks. I think maybe it would be prudent if
we went through each of these points one by one for discussion. So, let me just
rephrase it.
Point number one, what do you consider
to be the optimal spiking material and its preparation from the standpoint of
relevance to blood infectivity.
For example, SC 237 RML, would that be
a good metric for understanding infectivity in blood of a vCJD patient, for
example? Would a brain homogenate, as
opposed to a spleen homogenate, for example, be a suitable surrogate?
DR. JOHNSON: Glenn, haven't you already defined that it is
going to require brain by saying that you are going to drop it three to seven
logs? The only place you are going to
get those kinds of titers to shoot at is with brain, brain or spinal cord?
DR. TELLING: Any ideas of the titers of infectivity at
earlier stages during infection in spleen, for example? You are right. If you are looking for a six
log reduction, then brain would be the best.
DR. MANUELIDIS: I think 263K spleen is about five logs less
than brain, and I think in mouse models there is actually a higher infectivity
in spleen in many of the CJD models, sporadic CJD, and especially the Japanese
version of CJD.
So, they will actually be about 100 to
1,000-fold less than in brain, which is not bad, considering that for issues
after one strain it is about 109 infectious units per gram.
DR. TELLING: So, you could approach, with certain strains
and animal models, the levels of infectivity in the spleen, for example, that
might be required to demonstrate a significant log reduction in infectivity,
although brain is probably the better starting material.
DR. PRIOLA: Except that one of the considerations is that
the physical form of infectivity in the brain is going to be significantly
different than the blood.
So, this gets back to the aggregation
bit or whatever. So, using lower titered material probably wouldn't get around
that, because it is probably still in the same physical form.
What about the possibility of taking
advantage of Dr. Safar's observation that PRPSC binds to LDLs and
VLDLs, and possibly mixing those with brain homogenate, isolating those -- if
that is possible, i don't know anything about LDL biochemistry -- and then
using that as a spike, because that is something that normally circulates
through the blood. If you load it, then,
with PRPSE, maybe that would be physically more representative. Maybe not.
DR. MASTRIANNI: I am grappling with the terminology, optimal spiking
material versus what do we have available to do the job.
I mean, that is the question. What is
the best form of scrapie to use here. Brain material probably isn't the best
form.
I think as far as the LDL question
goes, that is a good idea and a good possibility if it is biologically
significant.
If we knew in vCJD patients, for
example, that we could separate, get scrapie from the LDL fractions, then that
would be a great starting material and, if it could be concentrated in that
way, it would be probably the best starting material.
The bottom line comes down to the fact
that we don't have vCJD patients' blood to be able to use as optimal spiking
material.
So, I think we are left with thinking
about brain for the question of the several logs of infectivity that we can
have as proof positive that we are doing something in the preparation, and the
question is whether we can do some preparation that closely assimilates what
might be seen in the blood, and specifically if we could characterize it or compare
it with an actual infectious model, maybe that shows that scrapie is contained
within the VLDLs. That would help.
MR. TELLING: I think probably the optimal spiking material
might be, if we are going to use brain, vCJD brain. The problem is that the
kinds of bioassays that would be required for sensitive detection of vCJD
infectivity are not as well developed, with the exception perhaps of the
bovinized transgenic mice, which appear to respond very well to variant CJD. So, that might be one avenue that we could
explore in the future.
MS. MANUELIDIS: I think the other thing is, there is the R3
mouse model, which apparently also is fairly susceptible, within 300 days, to
vCJD.
DR. TELLING: The R3 mouse?
DR. MANUELIDIS: Yes. That is even, I think, even across
species. So, in fact, one might consider using, for instance, sheep blood where
you can get a lot of it and then see if any of that is infectious. You have
huge amounts of blood that you can collect from a sheep with BSE, et cetera. I
just raise this as a possibility.
DR. TELLING: That probably falls under the endogenous
category rather than spiking, but yes.
DR. PRIOLA: I think you always have to go back to brain
homogenous just to start, because it is what we probably understand the most
about in terms of fractionating, infectivity, removing infectivity.
Another possibility -- and I think Dr.
Kreil brought this up -- are this GPI minus transgenic mouse model that was
created by Michael Oldstone and my boss, Chris Cheeseborough. They found lots of infectivity in the blood
using at least one strain of mouse scrapie.
DR. TELLING: 107 ID 50s per ml.
DR. PRIOLA: Yes, something like that. If that is the
case, whether or not it is in the correct physical form, I don't think we will
resolve that question any time soon for any form of infectivity, but there you
have got a form of infectivity in the blood that is transmissible.
That would be a spiking material that
could be used with current rodent model systems. It wouldn't be a terribly difficult
thing to do technically, I think. So,
that is another possibility. Of course, it is not variant CJD or BSE related.
DR. TELLING: Spiking and endogenous
model.
DR. PRIOLA: Exactly.
DR. TELLING: Any other points about
optimal spiking?
MS. KRANITZ: This isn't about optimum spiking. As the
consumer representative, I really don't qualify to delve into that. I can
listen and semi-understand, but I did want to make a couple of comments.
We have family members here in this
country who, in fact, two of them at present, one recently lost a loved one and
another one is actively dying of CJD, both of whom were born in the United
Kingdom and lived there the first, one 16 years, one 18 years.
So, we don't really know what we are
dealing with in this country. Obviously, because the United States has cut back
on their testing, their surveillance of animals, then the human surveillance
should be increased.
I think it would be important to
remember that we have an obligation to do everything we can to protect our
American population, considering that this enigmatic disease still has a great
deal of unknowns.
DR. TELLING: Thank you. So, I would like to move on to
point number two, selection of TSE strain and animal model. I think that the
two points are interrelated.
I think that probably we have
addressed as much as we can concerns relating to materials, optimum materials,
through use of spiking experiments.
Obviously, the models are in place,
including hamster infectivity and mouse adapted scrapie infectivity, and the
appropriate host would be the relevant ones.
One could imagine that BSE adapted
mouse infectivity, for example 301V, might be an appropriate TSE strain and the
appropriate mouse model that would be more in line with certainly BSE and
perhaps variant CJD. Any other comments along those lines?
DR. GESCHWIND: Just, I guess, to reiterate the point, the
importance of using both human CJD and also possibly using humanized mice.
I will give the example of the chimera
mouse Carston Court(?) has helped develop with the human mouse PRP. They were
greatly able to shorten the incubation period with just a two amino acid
substitution.
Unpublished but, with a third amino
acid substitution, they are now even further able to reduce it down to, I believe,
about 70 days.
Having a humanized model in human
would probably be the best model for human infectivity. At a minimum, I think that should be included
in any criteria.
DR. TELLING: Again, that was with
sporadic CJD. Okay, let's move on to
point number three, TSE immunoassays for PRP scrapie and bioassays for
infectivity.
I would like to open this for
discussion. Obviously, we have heard a lot about the CDI and its comparisons to
bioassay.
The comparisons appear to be robust
and reproducible, not just for CJD but also for chronic wasting disease and
BSE; correct?
That would obviously be a very
informative alternative to bioassay, and much more facile as an alternative.
DR. MANUELIDIS: I am making too many comments, but I do think
that there are caveats you have to really be aware of between that.
I think one of the things that hasn't
been done is, there are new tissue culture models, there are several models
that offer the potential to look at infectivity in a much more rapid way.
I haven't seen anything sort of
suggested on that or doing that, and I think that you are speaking about
something that might take 30 days versus, let's say, 300 days.
DR. TELLING: I think we are going to
hear from Dr. Soto tomorrow, but perhaps relevant to this discussion would
be the new techniques that are emerging for sensitive and rapid detection of
infectivity by in vitro amplification. Any thoughts about that or comments
along those lines?
DR. PRIOLA: I would say that, with all of these
techniques, they still have to be very, very carefully validated against the
bioassay.
I think that the PMCA, which is what
you were just referring to, and the CDI show great promise, but really need to
be carefully compared in the appropriate bioassay system at the highest level
of sensitivity that we can get, if we are going to switch over to something
that is sort of in vitro versus an in vitro diagnostic, especially for the low
levels of infectivitythat are in the blood.
DR. GESCHWIND: Just to follow up on that, I think that
certainly for right now, I think one important thing is that we need both the
bioassay and one of these other assays, that neither is going to be sufficient,
even if we do find that the CDI or other assays are even more sensitive.
It probably would still be very
important to show that you actually have transmissibility infectivity. So, I
think both are going to be necessary.
Dr. TELLING: Okay, finally, identification of
manufacturing processes that might alter TSE agent properties. We have heard
something along these lines this morning from Dr. Kreil. Any other thoughts or
comments about this?
DR. COLVIN: One thing I think we should keep in mind when
we think about this part of it, too, is what the numerator an the denominator
of the problem are, which is obviously very difficult to sort out.
If we are thinking about factor eight
and if we go back a decade or more -- probably more in this case -- in thinking
about what the story was with HIV transmission in terms of how many donors
there were in each lot of clotting factor that was used, initially the
manufacturers would say things like, it was from 6,000 donors. Ultimately we learned that it was probably
somewhere between 60,000 and 100,000 donors.
Now, if you take the UK sample that
Dr. Scott talked about earlier today, that there were three young people who
died or had tissue samples of unrelated causes out of 12,000.
If we are talking about a denominator
of what is in the clotting factor of 60,000 donors, then we would be thinking
about 15 or so people who would have potentially asymptomatic infection.
Now, we don't know if that is going to
be a contagious infection or contagious material, but I think we still need to
think that there may be some level in there that is higher than we actually are
anticipating, or people who potentially can transmit this disease.
DR. HAMILTON: I kind of have a follow up to what Dr. Colvin
was saying. Is there any data at all that specifies -- we know it can take up
to 38-1/2 years to show up, but is there any data at all that specifies how far
from actual symptomatology that the donor is contagious and could transfer that
through a donation? Is there anything at
all that shows -- I mean, three years, five years, 10 years? Do we know at all?
DR. TELLING: In terms of animal models, what we heard from
Dr. Brown last time was that halfway through the incubation period to the end,
to the manifestation of clinical symptoms, was the time at which titer could be
detected in blood. If anybody has any better recollection of that than me?
DR. CERVENAKOVA: My name is Larisa Cervenakova. I am from the
American Red Cross and I just would like to comment on these animal studies,
which actually showed that using the mouse model for the strain which is a human
derived strain and for variant CJD strain and for scrapie 263 strain in
hamsters, it seems that through half of the incubation period you have the
infectivity present in plasma and in the animals.
The pattern goes up to the clinical
stage of the disease, but there are some discrepancies in studies which were
performed by Claudio Soto's group when they compared the infectivity in the
blood of hamsters, and as they tried to correlate it with the data of PMCA. It
seems that, at a certain point close to the clinical stage of the disease, they
were not able to find the presence of a normal PRP in plasma of the animals,
and they were able again to detect it at the clinical stage.
DR. HAMILTON: So, in other words, that could be maybe why
some of it is not showing up in UK hemophilia patients? It just hasn't been
long enough?
DR. TELLING: I think the comments of Dr. Scott would
certainly underscore that.
DR. COLVIN: Something else that is relevant to this part
of the question, I am wondering, given the new data that we heard from Dr.
Safar today, or relatively new data, where does LDL and VLDL, as opposed to
some other things, fractionated in the fractionation of plasma, does it
fractionate with a cryoprecipitate or does it go somewhere else?
I think that would be kind of an
interesting question. I don't think we are ready to say, well, this means that
we are not going to see the TSE agents co-precipitate with factor VIII. On the other hand, it is something that I think
would be interesting to look into at this point.
DR. LEITMAN: It is not cryoprecipitatable.
DR. COLVIN: VLDL and LDL.
DR. LEITMAN: No, very few things are
cryoprecipitatable. Maybe someone from
PPTA could confirm that, but the two to four million dalton cholesterol
moieties are not cryoprecipitatable.
DR. KREIL: Really, obviously in preparation for that
meeting, knowing that Dr. Safar was going to share that newly recognized
association between VLDL and prions, we were trying to find information where
it would fractionate into.
The best information that we found was
actually from the early days of the Cohn fractionation. It seems that we are
going to fraction number three. That is
currently not being used for manufacture of further products. So, that would be
a waste fraction and therefore would be good news.
Again, this is very preliminary
information, certainly. It is something that we would want to take a look at.
DR. SAFAR: I think that is exactly the issue we are
looking into, where specifically the sCJD prions are. There are so many
fractions with spectrum of lipoproteins. So, the class that we identified is
pretty broad itself and it has sedimentation properties that are in the still
very broad range.
How each of those classes will behave
in any industrial production protocols or procedures, i don't think there are
sufficient data to even guess at this point.
I would agree with the comment that it
probably would go to the fraction three. There are additional aspects.
There are specific protocols,
plasmapheresis or lipophoresis protocols for removal of LDL from plasma, using
specific sized filters, which seems to be working for clinical cases with
hypercholesterolemia.
So, there is some technology and some
information for it, but it is not very precise, and it is definitely unrelated
to the CJD issue.
DR. TELLING: I think we will move on. There is a question
here.
DR. HOGAN: Before you move on to question 1-B, I just
wanted to comment, the very beginning of this in 1-A it asks about the
feasibility and scientific value of adopting standardized procedures.
I think we have heard from all these
people that we really don't know what the best spiking agent is, we don't know
the best model, we don't know how we are going to assess.
I think the idea of adopting at this
point would be a little premature.
Certainly this is all discussion for some time in the future, but I
don't see anything we can adopt right now.
DR. SALTER: Just to follow up on that point, we heard
from Dr. Kreil that the manufacturing processes are not standardized.
So, developing a standard for a
clearance study may not accurately reflect the flexibility that is needed, just
due to the processing itself. So, that is something that I think we need to remember.
DR. SCOTt: I want to thank you for these comments
because we do recognize how difficult it is to try to simulate blood borne
infectivity in a high enough titer to do clearance studies that would appear to
have meaningful levels of clearance. It
is also a fast moving science and we appreciate your input.
We did want to ask one additional
question, if anyone on the committee would like to comment, just related to the
first bullet that we had.
That is, does the committee see any
reason for a preference for using brain homogenate as opposed to microsomal
fraction or other fractions as a spiking agent.
DR. MANUELIDIS: Actually, I have a preference for using a
brain fraction. I think brain homogenate is so incredibly complex that I think
the most simplified fraction you have, even though it is not perfect, I think
it is something that is more likely to partition correctly in blood when it
goes into blood or into plasma.
The more garbage you have in it, I
think, the worse it is. There are many types of fractions -- noctosomal,
microsomal, et cetera, non-membrane fractions. I think those really are
preferable in many ways to a total brain homogenate.
I think also the kinds of conclusions
that people draw, it is very nice to have a western blot of brain homogenate
and say, well, this is PRP, but there are hundreds of things in that.
I think that sometimes one can be
misled to think that one is only dealing with changes in PRP where, in fact,
many things are being changed by the treatments.
DR. COLVIN: One thing I am not so sure about is this idea
that if we have some kind of a standard assay or a standard number of logs we
must reduce infectivity by, that that will stifle science.
I don't think that is ever the case.
Any time in science we do something, if somebody comes up with something
better, we are going to adopt that as well.
I just think at some point we need to
say, well, do we want to say four logs is adequate, five logs. Can we put a
number on it? The question is, what would be the spike that we would do it
with.
I agree, it seems like the products
are manufactured differently with different procedures and the like. However,
the idea, though, that because we have different products that means we can't
come up with some idea of what -- we just ignore the whole problem entirely
seems a little bit outlandish, in some way, to me.
I do think that, again, as soon as
somebody comes up with a good assay we are going to adopt it and people are not
going to stop trying to develop assays just because there is a standard.
We spend our time -- most of the time
I spend my time as an HIV person. For example, developing assays to look at the
effect of CCR5 antagonist, a new class of HIV drugs, sure we are trying very
hard to find standardized assays, knowing that it is not going to take long for
a better assay to be developed, but we want to have a standard now that we can
not just wait for even the best assay.
I don't think we are -- we are never
going to have the best assay. That is the nature of science. We keep moving
ahead and make things better.
As of September 2006, I feel like we
should come up with something that would say, well, we would like to say that
we are going to get a four log reduction and we are going to do it this way
but, as soon as somebody comes up with something better, we will use that one
instead.
DR. TELLING: I think we can probably
address that today, then. I think we can move on to question B, and we are
being asked to comment on the feasibility and scientific value of adopting
standardized endogenous study methods to assess TSE clearance in plasma derived
factor VIII.
There have been some recent
developments along these lines which have relevance. We have heard mention of a transgenic model
in which infectivity in blood is substantially higher, orders of magnitude
higher, which may hold some promise as an endogenous model.
Obviously sheep transfusion has been
another recent development that lends promise. It seems to me also that -- this
is obviously a personal preference -- possibly the use of infected cervids,
DUNL, for example, infected with chronic wasting disease, because of the
similarities between variant CJD, or at least some similarities between variant
CJD and CWD, that this might also be an endogenous infectivity model that would
be worth exploring. So, are there any
other comments from the committee members about question 1-B, endogenous
models?
DR. PRIOLA: Can you remind me, has anyone looked at mice
infected with variant CJD for blood infectivity? Has Larisa or someone done that with mouse
models of variant CJD or you?
DR. TELLING: I can't comment on that.
DR. CERVENAKOVA: The only study which was done, it was a study
using focuoca(?) strain. These were done together with Baxter.
I don't remember the numbers. There
was still some infectivity present in cryoprecipitate. I am really not prepared
with those numbers.
We didn't look through the clearance
steps. We didn't evaluate clearance using monoclonal antibodies with endogenous
blood, which is a very important step in the clearance procedure.
This was done with spiked material.
Thomas may add something to that. I don't know if they did some additional
studies using endogenous blood.
Until cryoprecipitation was done with
the endogenous blood and these particular steps, like depth filtration and
monoclonal antibody were done with spiked material.
DR. PRIOLA: So, there really aren't any models of
endogenous variant. The hamster model is the only one that has been relatively
well characterized.
DR. TELLING: Well, the closest might be BSE in sheep.
DR. PRIOLA: But that is very expensive to do. That is not
practical.
DR. MANUELIDIS: 301B in mice.
DR. PRIOLA: Is there infectivity?
DR. MANUELIDIS: I don't know. It is not from our group. I
think it is the English group did it.
DR. CERVENAKOVA: 301B, it was done by Debbie Taylor(?). She
found infectivity in the plasma of these mice, which was at the range which was
established for variant CJD mouse model, which we have in the lab.
Nothing more than that was done for
these particular models. If you talk about factor VIII, an endogenous
experiment was done with fucuoca strain in the mouse model up to
cryoprecipitate.
DR. MASTRIANNI: Were any of these models done by feeding or
by injections?
DR. CERVENAKOVA: No, everything was done where animals were
inoculated with intracerebral inoculation and blood was taken at the clinical
stage of the disease, and was processed that way.
DR. MASTRIANNI: It would seem that a better model would be
feeding the agent to an animal.
DR. TELLING: The animal model that probably comes high on
the list in terms of scientific value would be a primate model of variant CJD,
but this would have a lot feasibility, I think, at the moment. These models are
available, but certainly not widely available. Has that been addressed at all?
DR. CERVENAKOVA: Yes, it is too bad that Phil Brown is not
here for these discussions. As you may remember, on some of the advisory
committees data were presented on Baxter's experiment which was done using
squirrel monkeys.
It was a significant study actually
done in a way that squirrel monkeys were -- it was two phases, I believe. I am trying to recall some of the results
slides.
Some of the animals were inoculated
with sporadic CJD and variant CJD. Blood
from these animals was taken every three months during their lifetime, and this
blood was transfused into two squirrel monkeys. They always received this blood
from animals which were developing this disease.
The study was terminated, I believe,
five years after these transfusions. It means that during five years these
animals got multiple transfusions of blood from squirrel monkeys which were
inoculated with variant CJD or sporadic CJD.
None of the animals developed the
disease, but their organs, I believe are still under evaluation by the CJD
surveillance unit.
The other part of the study was that
blood from chimpanzees, which were infected with sporadic CJD, was taken and
separated into components, and these components were inoculated into squirrel
monkeys.
There was one transfusion from such a
chimpanzee which developed sporadic CJD. There was another part, when blood
from sporadic CJD patients and from variant CJD patients was separated into
components and inoculated into squirrel monkeys. I believe there were also no
transmissions.
DR. TELLING: Thank you. Any other information or comments?
DR. GESCHWIND: Just a comment and maybe you can comment on
this as well. I guess one concern I have about using any single animal model is
the differences.
One nice thing about using a cervid
model is the size and they are captive herds, but the transmission among the
cervids seems to be very different from what occurs in BSE as far as, for
instance, the horizontal transmission, all the herd getting infected where with
BSE, as I understand, it seems that the transmission seems to be from a single
source.
So, that is suggesting that there are
great differences in the way that these diseases are transmitted, and even with
cervids we are not sure about how the transmission occurs. Is it through urine,
is it through saliva, feces. Is it through something else?
I just think we have to keep that in
mind, that each species is different, and that one method probably won't be
sufficient.
DR. TELLING: Just to comment once
again, the difference in the BSE in the natural host, in bovids, is radically
different from its behavior in human beings. So, strain host combinations are
extremely important.
DR. PRIOLA: Just very quickly, I remember from the last
meeting this issue of multiple animal models came up.
I think the committee decided that
there should be multiple animal models used where possible, to test these
things, to take into account the differences.
DR. TELLING: We did actually go into that, didn't we. Okay, I am going to move on to question two.
I will just rephrase it. Based on the available scientific knowledge, please
would the committee discuss whether a minimum BSE agent reduction factor,
demonstrated using an exogenous spiking model and scaled down manufacturing
experiments might reasonably serve as an appropriate standard for demonstrating
TSE safety of the products. So, are we
comfortable with a four log reduction or a six log reduction? Does Dr. Epstein have something to say along
these lines?
DR. EPSTEIN: I wanted to ask the committee a question
based on the endogenous experiments, and maybe give people time to think about
question two.
Is there a feasibility to process a
large volume of blood from a large animal, sheep, goat, cattle, perform the
process but then attempt to concentrate the infectivity, so that you could do
read out in a rodent model.
The basic barrier in doing the
experiment entirely in the rodent is that you need thousands of rodents to look
for any residual infectivity.
You could process very large volumes
of plasma or whole blood from a large animal, if you could do the read out in a
small animal.
I just wonder if the committee members
could comment on the feasibility of available technologies to make concentrates
of residual infectivity from whatever end product one made.
In other words, if you made factor
VIII, could you concentrate the infectivity out of it and then read it out in a
rodent? Then these kinds of experiments
would be much more feasible.
Dr. TELLING: Not being an expert in blood concentration,
but certainly having some expertise in the animal models, I think what you are
proposing would be the way that I would want to do the experiment.
I would say it is extremely feasible
and extremely informative scientifically with the currently available,
sensitive and relatively rapid transgenic model for human, ovine, bovine at
UCSF and various other groups in the United Kingdom and various other locations
in the United States and Europe. I think those sorts of studies could very
readily be done and would be very informative.
DR. MANUELIDIS: I also agree, that is very nice
theoretically. Correct me if I am wrong. If you take a species like human, I
mean, you could actually concentrate human blood or bovine.
When you go across species, even to
the transgenic models -- and I have seen that with the BSE, I have looked at
the BSE and the vCJD models -- in fact, it takes 300 days. That is with a huge amount of material intracerebrally,
where you are going from brain to brain.
So, I really wonder if this --
although in principle I agree with you, I really wonder, with the current
models that we have, whether it will work.
DR. TELLING: Well, incubation times range depending on the
model and the host that the transgene is expressed, the level of expression,
there are a variety of different factors.
They range from anywhere as rapidly as
30 days as, you are right, 300 days. Certainly these various models are being
continuously improved.
DR. MANUELIDIS: I would like to know what the 30-day model
is. Would you get across species?
DR. TELLING: 30-day incubation time of SC237 in
over-expressing transgenic mice.
DR. MANUELIDIS: No, I am talking about across species from
the natural host. So, you take cow, bovine --
DR. TELLING: This would be hamster to mouse.
DR. MANUELIDIS: Bovine serum, sheep serum, that is infected
with cVJD, a cow brain, whatever you want, and you go into a mouse, any mouse.
All the stuff that I have seen in the
literature is basically 250 to 300 days even with the transgenic increased
susceptibility.
DR. TELLING: For the humanized model, the most rapid
incubation times that I have seen run about 100 days for the chimeric mouse
human transgene.
Another alternative would be -- this
is newly derived information -- that the Banglo looks to be a very promising
host for a variety of different models of infection, scrapie, CWD, not so much
BSE.
DR. SAFAR: The BSE in bovine transgenics, it is between
190 and 220 days for BSE intracerebrally. The issue in this case is significant
and I agree with Laura in this case.
For example, BSE in R3 mice titrates
into an end point only to 103 infectious units per gram. If you do
the same experiment in bovine transgenics, you will titrate 107 or
108 infectious units per gram from the same material.
So, the species barrier definitely
exists and it has to be considered in those experiments. So, I think there has
to be a list of I would call it relevancies.
First, the prion species or strain
relevant for that specific study, the clearance, decontamination, whatever.
Second, selection of the best host to
do that study. Third, because a lot of assays are based on the immunodetection,
it has to be also the sequence of PRP which is relevant for those assays which
are going to be used in the detection of PRP, if the PRP is going to be used as
a surrogate marker for infectivity.
In that case, every such assay has to
be correlated, as the biologists say, to prove in principle that that link,
that extrapolation from PRPSC levels that are infected, that it can
be done.
DR. KREIL: If I can offer another comment, beyond the
difficulties in read out, I think we should also keep in mind that it is rather
better understood that, for example, mouse plasma fractionates totally
different from human plasma.
I am not sure we know anything about
the fractionation of sheep plasma. So, even if we used that as a source of
endogenous infectivity and ran it through our manufacturing processes, I don't
think that would generate any meaningful information for understanding of how
that would translate into the fractionation of human plasma.
DR. TELLING: So, how do you feel about the spiking experiments,
then?
DR. KREIL: Well, at least there we know that the process
performs like it does in the large scale, which is why I pointed out that the
validation of the down scale is so important, because otherwise you don't
really know whether you generate information meaningful for the large scale
manufacturing process.
DR. CERVENAKOVA: I would like to make a comment on animal
models, and actually no one tried to do that, to use human plasma from sporadic
CJD case and tried to put it through the procedure, and inoculate it into
transgenic mice.
To my knowledge there are very limited
studies done in transgenic mice in general using human plasma. I don't remember
how many animals were inoculated at UCSF with human plasma from sporadic CJD
cases, but it was just six or eight animals inoculated, R3 strain, inoculated
with variant CJD.
From this number of animals you
definitely may not get any results. In my experience if you take a grouped of
five animals and a group of 20 animals and you inoculate those five animals and
let them sit and you inoculate 20 animals and let them sit as a separate group,
you will not see any infections in the group of five animals.
I believe that, if infectivity is
present in the blood of variant CJD patients and there are some indications at
least that abnormal PRP might be present there, we should be able, by
concentration steps, to concentrate the infectivity and, by inoculation of
transgenic mice, which carry human PRP gene and have a short incubation period,
we would be able to see it, if it is there.
With variant CJD, it is a very
different matter because, even so, even humanized transgenic mice, they do show
sensitivity to the variant CJD, but the incubation period is so long, that we
are not able to see low levels of infectivity which are present in blood.
This is true for bovinized mice, I
call them, because they also will have a very long incubation period and we
will be not able to see the infection if it is present in very low titer.
DR. TELLING: So, we are lacking a host readout for variant
CJD, even if w could fractionate from variant CJD infected blood.
DR. CERVENAKOVA: I believe so. We need to look for a good
model for variant CJD for low levels of infectivity. I also am wondering -- I
know that no one did it, actually, and we are planning to do the studies to try
to use transgenic mice and blood from transgenic mice to prove if we can
transmit the disease by blood, inoculate transgenic animals in between.
DR. GAYLOR: On question two, I think it is rather
difficult to answer question two at this time without having the FDA risk
assessment in front of us.
I had the advantage of reviewing the
factor VIII risk assessment as of about a month ago. I don't know how much it
has changed in the past month.
It is true that the most uncertain
part of that estimation is the manufacturing reduction factor. That is the
largest source of uncertainty.
You can assume various log reduction
factors and estimate risk, but those assume factors. It would appear with, say,
a log 6 reduction that I guess the best term to use is you come up with a
negligible risk without defining negligible mathematically.
If the risk is -- if the reduction
factor is only, say, log 3, you still may come up with a negligible risk. So,
it is kind of hard to say whether log 3 or log 6 is what is needed. A log 3
reduction may be adequate. I don't think we can really answer that until we have
the benefit of looking at the risk assessment at the next meeting.
DR. TELLING: Could we defer this question until that
time? I think we will do that then. I am sorry, there is another comment here.
DR. POWELL: It may be the committee's decision to defer
the question of the specific value, but in terms of some comments that FDA
might consider as they think about that, I think one thing that is important to
consider is how a log reduction is defined.
The nominal log reductions that I have
heard talked about I presume to be average values and average log reduction
achieved in one or more replicates.
DR. SCOTt: That is correct. So, you have a starting
absolute amount of infectivity, which is the volume of material that you have
times the amount of infectivity per ml.
Then you have your manufacturing
process. You will probably come out with different volumes at the other end.
So, you calculate that absolute amount of infectivity and you subtract it from
your starting material.
DR. POWELL: When we speak about a four log reduction
factor, I think it is important to consider not only the nominal value or the
mean value, but also whether there is some variability in the performance that
you might detect through replicates under the same conditions.
For example, you might have a process
-- two processes -- that both achieve an average log reduction of whatever --
four -- but the one that has the greater variance in terms of its performance
is going to result in a greater likelihood of high levels of residual
contamination.
So, it is not simply the average
performance but also the variability in performance that is important to
consider when you are formulating a performance standard for a log reduction of
whatever process.
For example, it might be formulated as
95 percent confidence of no less than X log reduction as opposed to a more
ambiguous nominal log reduction.
DR. SCOTT: That is exactly correct. Because each
manufacturing run has slight differences in reality in terms of the exact
amount of alcohol, the precise pH, there is some variation, especially when you
are talking about precipitation studies, which are not generally considered, if
you will, robust.
In viral validation studies, it is
possible -- because you can have a fairly high throughput of number of
experiments -- to look at robustness and see how much the variations in the
manufacturing ranges can impact that, and come up with a plus or minus this and
a confidence interval.
That is a lot more difficult in these
studies if you are depending upon assays like bioassays that take a long period
of time.
It is not impossible, but you are
absolutely right. There is a lot of information in the literature of replicate
studies.
In general, it would be possible, I
think, for us to come up with how much range there is in some of these studies
as published that is useful.
DR. BROOKMEYER: This question is posed as if there is a
single reduction factor to be aiming for. The reduction factor could also
depend on what the input is that you start with.
If you start with a lot you might have
a big reduction, where if you start with very low amounts, you may not have
much of a reduction factor.
So, there is going to be some swing
here in the reduction factor depending upon what your baseline is and what you
are starting with.
My question is, is that right, and is
there any data to reflect on that, on how the reduction factor could vary with
what your initial input is?
Wouldn't that be a critical question,
actually, for the risk assessment and for assessing your absolute risk here?
DR. POWELL: You are speaking about a concentration
dependent effect of the performance of a process as opposed to an incoming load
variant, specifically the concentration dependency of the process in terms of
its efficacy.
DR. BROOKMEYER: That was the question I had, too, in looking
at the literature, because we see this in antibody antigen dynamics, that there
is a dependency. I am wondering, in some
of these precipitation sorts of processes, there might be a similar sort of
effect.
DR. MASTRIANNI: I had exactly that same question. Along the
same lines as Dr. Kreil was speaking about, log reduction factors, he was
adding them up and I wonder if that is a reasonable thing to do also. Maybe you
can comment on that. When you are adding
logs, you are actually multiplying factors; right?
DR. KREIL: Well, obviously that is something that needs
to be demonstrated and I guess the agency's terminology has been where steps
can be demonstrated to be orthoganol, so different in terms of mechanism of
action, then it is appropriate to add them up. If that cannot be demonstrated,
then it would not be.
DR. MASTRIANNI: Do you have any data to suggest that if you
start with a higher titer of infectivity the log reduction is different than if
you started with a lower titer of infectivity?
DR. KREIL: I would not be aware of nay such data. That
would assume that the mechanism would be a saturable mechanism, where the
partitioning is not.
DR. PIFAT: I had a comment. I am from Telecris. We have
conducted experiments where we spiked plasma and carried the manufacturing
process almost to completion and have shown clearance over the entire process,
and have been able to show that actually small levels of clearance, one or two
logs, that are contributed by individual steps actually add up when you conduct
the experiment from start to finish.
DR. SCOTT: It is the empirical way to determine whether
steps are additive or not, and it would seem the most valid way to do it is
actually to do the steps both separately and in sequence.
There are some published examples
where those either have or have not been additive. It is a little hard to
predict what is going to happen without doing the experiment.
DR. POWELL: One other issue regarding things that FDA
might consider in terms of a performance standard, is that we are dealing with
a continuous unit as opposed to a discrete unit.
Typically, in microbial risk analysis
when we think about a log reduction, say, down to a mean dose of -- well, let's
say we start with four logs and apply a four log reduction factor. We have a
mean dose of one discrete unit per dose, because that is a discrete, countable
colony-forming unit or what have you.
You have -- if you assume, say, a
puissant process, there would be, oh, probably about a 40 percent probability
that any particular dose that you selected from that lot would be absolutely
free of any infectivity.
That is not the case with an ID50,
which is a continuous variable. So, applying the sorts of procedures for your
highest reasonable concentration plus a two log safety factor is not, I think,
directly relevant when you are dealing with a continuous unit like an ID 50, as
opposed to a discrete unit, like a colony forming unit in microbial risk
analysis.
DR. GESCHWIND: Maybe this is more of a question, but sort of
the way I think of this is not so much log reduction, but we would want to say
that you have reduced the infectivity to below one infectious dose. That would
seem to be it. I am wondering how that
fits with what you are saying.
DR. POWELL: Again, your unit is not one percentile. It is
an ID 50. It is the median infectious dose.
So, one ID unit is actually the dose
that would make 50 percent become infected. So, the unit is quite different
than the countable units that we typically deal with when we are dealing with
virus or infectious bacteria.
DR. GESCHWIND: So, just to follow up with that, then, is
there a solution to that? I think that
is the reality of what we are dealing with. Are we going to have to get to the
point where there is never an animal that gets infected, which would be zero
ID?
DR. POWELL: It would be a function of your desired level
of protection. Simply from the calculation perspective, it would be
inappropriate to use the sort of procedure that you would use for bacteria and
apply it to an ID 50, which is a continuous unit.
DR. MANUELIDIS: I actually have a question about that. In
virology, as I understand it, most of the viruses are usually multiple.
In other words, even if you do a
colony count, there is a certain efficiency. Maybe the one exception might be
polio virus, which can have a one to one article to, let's say, colony change.
Virtually most of the other viruses
are like 100 to one or something in that kind of range. Maybe you want to sort
of clarify how you would -- I think it is fair enough -- one ID 50 is
essentially saying, well, you want to be something like at least 10-fold under
that, I think, if you want to say it is clearance.
I assume that the infectivity,
particle infectivity, is relatively low in these diseases, as it takes a long
time to get, but I may be totally wrong, but I would say something 10-fold.
DR. POWELL: Similarly, with bacteria, we can count
colonies. We don't know how many bacteria are members of a particular colony,
but this is more akin to -- an ID 50 would be more akin to -- an ambient
concentration of a chemical pollutant.
Obviously, at some level we are
getting down to a fraction of an ID 50 that constitutes a countable infectious
unit.
The idea that the two log safety
factor that is kind of a convention in microbial risk analysis could be
directly translated over to dealing with ID 50s, I think that FDA needs to
consider that.
DR. TeLLING: Well, I think these comments are probably
very useful to FDA. We will move on to question three, which is somewhat
related.
So, the question is, considering the
outcome of discussions on question two, notwithstanding the fact that we are
going to just defer this, in cases where only lower levels of clearance can be
demonstrated for a plasma derived factor VIII, should FDA consider the
following:
A, labeling that would differentiate
the lower clearance products from other products with sufficient TSE clearance;
B, recommending addition of TSE clearance steps to the manufacturing method; C,
performance of TSE clearance experiments using endogenous infectivity models;
D, any other actions.
DR. GAYLOR: Again, since three depends on two and we have
deferred two, it is a little hard to -- we could still discuss three a bit, but
it is kind of hard to come to any conclusions on three without concluding on
two.
DR. TELLING: I wonder if the committee had any thoughts
relevant to this that would be useful for FDA to hear.
DR. LEITMAN: I am a little concerned about A. Labeling is
something that we do for the public. So, labeling something as differentiating
it as being less good than something else is a concern.
It raises anxiety on behalf of the
public. We are talking about negligible risk to begin with right now. I don't
think any labeling should be discussed at this point.
DR. JOHNSON: I was also going to question about A. Is this
considering a required labeling saying, in the standard tests, this material is
better than X,Y,Z but not as good as P,Q, or is it allowing labeling,
permission to label? What is FDA's
position on that?
DR. TELLING: FDA?
DR. GESCHWIND: Maybe toward this, if FDA could also comment
about products that have already been labeled that were alluded to in the
handout?
DR. EPSTEIN: Our current policy is that we have permitted
voluntary labeling of clearance when we think we have seen data that are robust
and high quality.
We have not allowed those labels to
further assert any greater assurance of safety, only to assure the public that
there are these clearance experiments and here is what they show.
I think at the present time we are not
talking about mandatory labeling, but if we thought it was a safety issue, then
potentially it could be mandatory.
Right now, what we are talking about
is labeling related to clearance with some uncertainty whether that is linked
to a level of safety assurance.
The point of that, why we have allowed
that at all, is that we wanted to provide an incentive for companies to do the
kinds of experiments that you have been hearing about. So, that is not a clean answer, Dr. Johnson.
DR. GESCHWIND: I just think that it is important, related to
that, that the market place is going to drive the better product.
If there are tests or if there are
methodologies out there that do provide higher clearance, that is something
that consumers might want, of course albeit with the caveat that clearance may
not be infectivity. That is what Dr. Epstein alluded to.
DR. TELLING: I am sorry, what was the last?
DR. GESCHWIND: That just because you have shown clearance
doesn't mean that it is necessarily a safer product.
MR. BIAS: After this decision is made, most of us will
go home and life won't be any different. If you are a patient who infuses his
product, every company that has that advantage of removing that many more logs
-- I remind the committee that we haven't seen any infections related to plasma
based products in many, many years -- those are the people who are going to get
that message.
It is going to show up at their door.
It is going to show up from the people who deliver their products. It is going
to show up at every hemophilia meeting they go to.
It is going to be used as a strong
marketing tool to separate one product from another, and there are some
complications there.
Some of these products are true plasma
products. Some of them have von Willebrand factor in them, which is essential
to patients. We are finding more and more patients who have hemophilia and some
sort of von Willebrand factor missing and need products that have both strong
hemophilia and some von Willebrand factor in them.
It is a much more complicated issue
than allowing the manufacturers to claim an advantage through labeling that
really turns into a strong marketing pull.
We have, among the recombinant
manufacturers, we are finding in local communities now that they are pulling
together small groups of consumers in private situations without physicians
present, and pitching their products as if one is substantially better than the
other.
Well, we have seen no infectivity or
transmission in any of the products, no differences whatever. It takes the
decision out of the physician and the patient's hands, and makes it into a
marketing tool for the manufacturer.
So, at this time, I couldn't support
any labeling change based on lower clearance, because the products are so
unique to the patient's needs. It just
seems inappropriate at this time with so many unknowns, in my opinion.
DR. SEJVAR: I guess just to build on that, if the labeling
is for the benefit of the consuming public, I am not so sure how much of a
service there is to make these statements in the absence of any clear idea of
what the biological relevance is. I
guess I feel a bit hesitant about the whole labeling issue as well.
DR. MASTRIANNI: I guess one could imagine just a standard
label that says, meets criteria for FDA requirements of being prion free or
something to that effect, that all things are labeled with a certain standard
to suggest that either risk from infection is very low based on the product
and/or has met some criteria which do come from this meeting as far as standard
levels of reduction of infectivity.
In other words, just normalize it or
equalize it for all the different companies, just create a standard rather than
differences in higher or lower levels of standard.
DR. TELLING: Let me ask if FDA is satisfied with the
comments that have been presented to them so far. I think that while there is
some uncertainty -- a lot of uncertainty -- there has been some constructive
discussion here. Would that be fair to
say?
DR. SCOTT: It has been very useful, but if committee
members have comments on B or C, and let's just take a hypothetical that, at
some point, the committee or we felt that there wasn't a sufficient level of
clearance, what do you think about options B and C, and are there any other
actions to consider in that scenario?
DR. PRIOLA: With regard to C, thinking about what Dr.
Epstein had asked about large animal models or in concentrating, and what Dr,
Kreil said about the fact that blood from these models does not fractionate in
the same way as human blood, it would seem that, while it is feasible to use
some of these rodent models of endogenous infectivity, the scientific value
might be limited, given that they don't fractionate in the same way as human
blood.
So, it might be that performance to
TSE clearance experiments using endogenous infectivity models, as in C and I
guess 1B, might not be so feasible scientifically if they don't mimic the way
human blood fractionates.
DR. HAMILTON: Regarding B, I think we are always
recommending -- and manufacturers know that -- the committee is always
recommending do whatever you can to make the product safer and as safe as
possible.
I would have to agree with what Mr.
Bias said, to some extent. Unless a product is really, really, really better in
this line, that we be very careful about what we say about the products without
enough substantiation for that.
Like you said, it is very difficult to
convey to the consumer, I think, adequately without fear of what the risk is.
Clearly, from the discussion today, we
don't really know yet what the risk is and, without being able to explain that
to the patients in a way that they can understand, I would be very leery of any
current extra labeling.
DR. TELLING: So, I think we would like some more detailed
and thorough assessment of the risk, maybe in the next meeting. Are there any
other comments? Dr. Scott, before we adjourn?
DR. SCOTT: If there aren't any other comments, I would
like to thank the committee and assure them that we are planning to present the
risk assessment at some future date, and we appreciate all your thoughts that
have gone into these questions. You will probably be hearing some version of
them again.
DR. TELLING: Okay, thank you very
much. Let's adjourn for a 15-minute break and we will reconvene at just after
2:45. Thank you.
[Brief recess.]
DR. TELLING: So, we are going to get back to committee
updates. Dr. Weinstein, from the FDA, is going to update us on the status of
FDA's initiative on communication of the potential exposure to vCJD risk, and
an investigational product, plasma-derived factor XI that was manufactured from
United Kingdom donor plasma.
Agenda Item: Committee Updates: Status of FDA Initiation on Communication of
Potential Exposure.
DR. WEINSTEIN: As many of you know,
there is a possible but as yet unproven risk to approximately 50 individuals
who were involved in investigational study of a product, a factor XI product,
that is used to prevent or treat bleeding in these patients who are deficient
in factor XI.
The factor XI was made using plasma
from donors in the United Kingdom where the human disease variant CJD has
occurred.
Now, it is important to note that the
factor XI product was not made from the plasma of anyone known to have
developed the disease, and that no one who has received this product is known
to have become infected from it.
Now, although the product was not made
from a plasma from anyone who developed variant CJD, it is still possible that
someone using the factor XI product could have been exposed to the agent
because of a donor who felt well at the time of blood donation, who was
actually carrying the variant CJD agent.
Now, FDA has made a computer model of
a risk assessment, and we have presented this risk assessment to you in open
session in February 2005, and we also received comments about it and
information when we were discussing the factor VIII model in October of 2005.
We have taken those comments and have revised the risk assessment.
One of the important elements of your
recommendations was to advise the FDA to consult with SGEs -- that is special
government employees -- including members of the hemophilia community, to
obtain advice on risk assessment, on interpretation of the assessment and on
its communication.
Again, by way of background, the
factor XI was manufactured in the United Kingdom from UK plasma, and was used
by approximately 50 patients between 1989 and 2000.
Most of the cases of variant CJD have
occurred in the United Kingdom. We heard today that 162 out of 196 reported
cases have occurred in the United Kingdom.
Individuals in the United Kingdom are
at higher risk for the disease than individuals elsewhere because of the
greater potential for food chain exposure.
In the United Kingdom, health
authorities have notified recipients of plasma derived coagulation products,
which were of course made from UK donor plasma, that they might have an increased
risk of variant CJD in addition to the risk of eating contaminated beef
products.
In CBER's risk assessment model, which
again you have had an opportunity to comment upon, we found that the most
important factors affecting the risk are the clearance of the variant CJD
agents through manufacturing, how much of the product individuals use, and the
prevalence of variant CJD in the United Kingdom donor population.
Now, we have completed the risk
assessment and we have distributed it to SGEs, to the hemophilia community. We
are waiting for their comments, which we anticipate getting very soon and, once
we have those comments, FDA will present the risk assessment, an interpretive
summary of the risk assessment, and advice to patients.
We intent to submit this risk
assessment and discuss it with the IND holders of the factor XI products to
obtain their comments and to answer questions about the risk assessment.
We will strongly suggest that the IND
holders contact and advise patients, the subjects who participated in this
study, and there will be a public posting of the risk assessment and
notification to hemophilia organizations of the risk information.
We will have a follow up among FDA and
IND holders to assess the impact of the message, the number of patients
contacted, and the need for additional information or assistance from the FDA,
and for the Centers for Disease Control and Prevention. Thank you.
DR. TELLING: Thank you, Dr. Weinstein.
Are there any questions or comments?
MR. BIAS: I am curious. I notice the different points
of communication here and that is pretty good, but from that list, I don't see
any communication to physicians.
The way hemophilia is treated, as you
know, is through a network of federally funded hemophilia treatment centers,
and it would be good for them to know. I know you have a plan to educate them.
The most difficult part about treating
hemophilia is the additional providers who fall outside the hemophilia
treatment center team. Those would be internists and dentists and others who
treat what turn out to be complications of hemophilia.
Almost every patient has bleeding into
their dental area, if it is not well controlled. Those are the physicians, especially those
physicians who are treating our medicare and medicaid patients, that I am most
concerned about getting some kind of formal communication from the FDA.
Those are the physicians that are
going to be most likely. They are already underfunded for treating these
patients. Those are going to be the physicians that are most likely to walk
away from treating them.
So, I don't know if it is so much of a
question as it is a plea that some kind of -- that we develop some kind of
communication specifically for that physician, and that it be distributed
through medicare or medicaid communication lines, so that there is some kind of
pre-communication to them.
There are going to be two kinds of
confusion that come out of any kind of announcement. One, they are going to see
hemophilia and not recognize XI, which is a very small percentage of the
population.
The second is they are going to see
hemophilia and say, you know, I don't get paid for this patient anyway. So, why
should I continue to treat them, especially if I am putting my practice at risk
on some level. So, that would be my
advice, comment input.
DR. WEINSTEIN: I appreciate that. I do point out, of course
that we are, first of all, concentrating on the interaction between the IND
holder and the specific factor XI patient, targeting this very small, hopefully
well defined population here, but your comments are well made here.
There will be comments about the
variant CJD and hemophilia treatment, factor VIII directly related to the
potential variant CJD in factor VIII products.
Again, this would be an issue that we would bring forth to the committee
at a later time.
MR. BIAS: And it all could go very smoothly, unless it
is a slow news day, and then we could see it 15 times over every news network,
and it could be a hemophilia crisis because they are not going to get it right.
They are not going to indicate that it is a small group of patients.
They are going to say, hemophiliacs,
hemophilia on the ticker tape. It is going to be the story of the day,
depending on what is going on. So, just keep that in mind as the communication
plan moves forward.
DR. HAMILTON: I would just endorse that, but add a PS to it
and say that, while medicare and medicaid are probably going to be the first
physicians to react in that way, because all models of reimbursement follow
medicare and medicaid, it is going to go to the private pay sector as well.
It ranges from pediatrics to
orthopedics to everything, every different area of medicine that treats a
patient like that.
I realize that you are going to start
out with the small cadre of patients with the IND and the IND holders and so
forth, but as Val said, if it is a slow news day and word leaks out, then you
are going to have all these other doctors talking about it, the patients are
going to be talking about it.
Somebody is going to say to a patient,
well, I heard, you know, and then here we go and we have a mass communications
hysteria on our hands.
DR. COLVIN: Just a question about how sort of factor XI
was used. How may donors' plasma was used to manufacture the factor XI, just
out of curiosity. Was it similar on scale to how many donors are used to make
factor VIII?
DR. WEINSTEIN: We estimate -- we described the model
earlier. I think it was about 20,000 donors per pool.
DR. COLVIN: Is the pool a realistic one, or is that the
industry based estimate that is a factor of 10 off?
DR. WEINSTEIN: It is pretty accurate.
DR. COLVIN: Then was factor XI used in an ongoing way,
sort of like factor VIII is used?
DR. WEINSTEIN: It is not used in the regular chronic
fashion. It is used mostly in the preventive field for occasional bleeding.
Factor XI deficiency is much different.
DR. COLVIN: It is a much more mild disease. Has the IND
been approved for this?
DR. WEINSTEIN: There are several INDs that were used between
1989 and 2000. It is not a problem in that sense now.
DR. TELLING: Thank you, Dr. Weinstein. So, next we are
going to hear from Dr. David Asher. He is going to summarize discussions of the
World Health organization relating to distribution of infectivity in tissues of
animals and humans with TSE.
Agenda Item: Summary of WHO Consultation on Distribution
of Infectivity in Tissues.
DR. ASHER: I can't really summarize in 20 minutes a
proceeding that took two and a half days, produced a 53-page document, small
print, that just issued, and involved presentations by 42 speakers, but I will
do the best that I can under the circumstances.
The consultation is one of a series
that was organized by Anna Padilla of the WHO secretariat over the past number
of years. She is aided by others in her
section, but she really is the prime mover.
The single most important thing I can
hope to show you is that the document is readily available, either directly
through this PDF that is on the little handout, or through a link in the WHO
web page.
In the handout, what I have done is to
put in the slides condensed a little bit, that include things that were
actually covered in the consultation.
I will show some other material that
is explanatory, because it is very hard to follow the reason for the
consultation, I think, without either reading the whole document or with a
little explanation.
The purposes of the consultation were
several. Specifically, the goal was to revise a 2003 consultation document on
the safety of medicinal products, particularly biological products, with
emphasis on vaccines and products derived from human blood, with a little
attention to human cell tissues and products derived from them.
To support that, scientific
information, particularly during the past two years, was reviewed, risk
assessments provided to regulatory authorities in several countries.
One important goal of the consultation
was to provide advice to national regulatory authorities with limited
resources.
The WHO in general does not expect
that its consultations will be particularly -- I don't mean to say valuable,
but they don't have the same weight in countries that have elaborate approaches
to the TSEs, like the United States, Canada, the European Union, Japan,
Australia and New Zealand.
They also don't expect to get
underdeveloped countries are going to be able to undertake some of the steps
that we have taken in our countries, but they do believe that, for reasons that
I will show you in a second, that the countries that don't have the resources
that we do could nonetheless be aware of the issues because of the nature of
the risk.
A special goal that we will end with,
and you will hear a little more about, I am sure, during the open public
hearing, was to summarize the available information about the distribution of
infectivity and abnormal prion protein in tissues and body fluids in the TSEs
that the WHO deemed most important, which are Creutzfeldt Jakob disease in
humans and the natural diseases of ruminants.
There has been no attempt made to
summarize the huge body of information on rodent models, more because it has
been a North American problem, was chronic wasting disease, or mink
encephalopathy, for that matter addressed.
There was an ambitious agenda
summarized in the document. I won't go through everything, although I made
three or four slides that summarized points that seemed to me to be of
particular interest. I will go through
briefly the TSE infectivity table at the end.
The reason for the concern is the
continuing recognition of BSE and variant CJD in countries throughout the
world.
There are now 26 countries that have
recognized BSE in native cattle and, although the epidemics do seem to be
declining, two countries, two new countries, recognized cases this year.
The United Kingdom, which has had more
than 184,000 cases, still had 225 cases last year, and cases have occurred, as
you know, in North America. We hope that the cases in Canada are going to turn
out to be largely restricted to western Canada. As far as I know, there have
been no cases in Ontario, Quebec or the Maritimes, but it bears watching.
Another risk is that caused by the
export of contaminated meat and bone meal from the United Kingdom during the
years of concern throughout the world.
This map is based on UK customs and
excise records that show that contaminated meat and bone meal is at least on
record as having been exported to a number of countries. It wasn't exported,
some small amount, by the way, to the United States and Canada, but also to
countries that haven't recognized BSE, like the former Soviet Union, Indonesia,
Thailand, and one doesn't know what those countries did with the meat and bone
meal. There is some possibility that
they introduced BSE and have not recognized it
Another risk comes from travelers. We
know that people have been infected with variant CJD n the United Kingdom or
perhaps other countries, and then returned to their homes bringing the
infection with them, and the transfusion risk presumably travels with them to
their home countries.
So, it was felt that this disease has
become a global issue, and all countries should at least be aware that the
disease exists.
Now, how does it compare with AIDS and
tuberculosis and malaria and avian flu and automobile accidents and all the
major killers?
Obviously, many of these countries are
not going to be able to do much in the great scheme of things, are not going to
be able to invest in defending themselves against a relatively remote risk, but
at least they should be aware of it.
Also of concern, it wasn't this goat,
but there has been recognition in the past couple of years that BSE, at least
as defined by recent profiles of putting extracts into mice, BSE has not only
been transmitted experimentally to small ruminants like the sheep and goats,
but there is now evidence that one goat in France appears to have been infected
and possibly one in Scotland.
that mens that contaminated feed
presumably got used in Europe to feed these animals, and in many parts of the
world small ruminants are a much more common food source than are cattle.
The natural history of this disease in
small ruminants seems to be quite different than that in cattle. The two
phrases that got amputated from the bottom of one of the slides in the handout
that referred to the transmission of BSE to sheep and goats.
Also, there remains a concern about
variant CJD. Some of that was explained by Dot Scott earlier today. Although
the clinical disease has peaked in the United Kingdom about eight years after
the peak of BSE in cattle in the United Kingdom, the finding of the two
positive appendices suggests at least the possibility of the prevalence of
undiagnosed cases there may be higher than was expected, and one doesn't know
how long before the appendix becomes positive the blood becomes positive.
So, there is an unknown blood risk in
the BSE countries that is of concern, and the three transfusion transmitted
infections, the first one recognized in December 2003, only increased that
concern.
The growing number, relatively rapidly
growing number of cases in France, 17 of whom had no substantial history of
time spent in the United Kingdom, is another source of concern, as is the
Japanese cases, that Dot mentioned, who spent less than a month in the United
Kingdom.
There is never any reason from animal
studies to doubt that a single exposure might, under adverse conditions, be
enough to infect a person, but it reminds us that there is a residual risk that
we haven't dealt with by deferral factors.
So, those are the reasons why the
World Health Organization remains concerned, hosts these consultations. It is
gratifying that, except for blood, no other new class of medical products has
been recognized as transmitting CJD in the past 10 years, but here have been
now over 360 recognized iatrogenic transmissions.
One of the things that was attempted
at the consultation was to simplify, for those of us who are more public health
oriented, the nomenclature regarding abnormal forms of the prion protein.
Jiri Safar discussed this this
morning, the various forms of abnormal prion protein protease resistant,
protease sensitive, degradation products of different lengths.
We didn't get into the five or maybe
six subclasses of prion proteins in the Cohen classification scheme or the
four, maybe it is five now, in the Parker Gambetti scheme. It became so
difficult just to talk to each other that it was proposed that, for our
purposes, the term PRPTSE be
used for all the abnormal forms of prion proteins, regardless of their
molecular nature, and let the specialists, in the end of days, figure out what
those forms were.
So, for our purposes, we used that
instead of PRPSC or PRPRES, although UK authorities, before the end
of the meeting, had started referring to PRPD for disease related PRP. So, go
figure. At least for the rest of this
talk, we will use PRPTSE .
I went through the proceedings and I
selected some points that I thought were of particular interest to this group
today, and I list them on the next three or four slides:
Naturally affected cattle, infectivity
detected by mouse assay demonstrated
only in brain, spinal cord, retina and a pool of nictitating membranes, but not
in pools of lymph nodes.
Infectivity was detected in some
peripheral nerves, and a solitary muscle of a single case of BSE in a German
cow. That is a greater concern in Europe where there is more BSE than the
maximum of one case per million, we hope, that USDA estimates here. There was
only infectivity found in a semi-tendonoisis muscle using only an extremely
sensitive transgenic mouse that Martin Groship(?) has developed.
Still, it would be disturbing if meat
itself was intrinsically infected rather than neural tissue, because
contamination has been the thing that is of greatest concern for the safety of
the food supply.
Cattle experimentally exposed to oral
route BSE infectivity detected in distal ilium throughout the course of disease
after six months in the palatine tonsil, and only in cattle assay,
demonstrating that the mouse assay, except for some transgenic mice, is
considerably less sensitive, and this is the part that got amputated from your
handout, BSE experimentally transmitted via the oral route to sheep and goats
and one, possibly two, goats now recognized in Europe to have been infected
under field conditions.
No sheep has been recognized, but they
both got fed similar, presumably some similar feeds having presumably some
level of contamination.
Under specific experimental
conditions, brains of some TSE infected rodents may be infectious by bioassay,
while TSE remains undetected. That problem has been mentioned here today.
Immunoassays detected PRPTSE in brain of BSE in cattle at least thee
months before onset of illness. No immunological method yet validated as
sufficiently sensitive to detect PRPTSE in the blood of infected animals or humans,
although promising initial results were reported by several groups of
investigators and we will be hearing tomorrow reports by all six of those
groups who reported last year, plus a report by Dr. safar.
Transfusion experiments have not been
conducted in cattle, although studies using small amounts of blood and spleen
of cattle assayed in mice and cattle, in operating cerebrally, failed to detect
infectivity.
The regulatory conclusion to that, a
conservative regulatory approach would assume that bovine serum might
potentially contain TSE infectivity, presumably, in small amounts.
Ruminant blood, blood derivatives such
as fetal calf serum in cell culture, and bovine serum albumen have not been
identified as a source of infection, but should be properly collected to reduce
the risk.
However, blood of sheep with both
experimental BSE and natural scrapie can be infectious and, because scrapie and
BSE agents behave similarly in sheep and goals, the blood of small ruminants
should either be avoided in preparing biologicals, or selected very carefully.
I think because sheep and goat blood
are used as a source of immune sera, some quite useful sera, the second
criteria is more feasible, at least on a worldwide basis.
There is a continuing need to ensure
that all regulatory authorities with limited resources have ready access to
reliable and up to date information when assessing TSE risks and evaluating
medicine product safety. Those were one
of the main reasons for the consultation.
Although I am not going to review the
42 talks, I think that I found comparisons of the results of the natural risk
assessments for variant CJD in blood to be informative, because they seemed to
be, although independent in their design, they seemed to come up with very
similar results.
For example, Marc Turner presented an
assessment for the United Kingdom suggesting that one in 120,000 transfusions
there might be from donors incubating variant CJD.
If strict indications for transfusions
were observed, the benefits clearly exceed the risk. One of the take home
messages is, as in many other areas of medicine, if you don't need to use a
product, don't, and then there won't be any risk assessment associated with it.
Plasma derived products, their
estimates suggest that minimal risks, surgical and dental instruments, human
cell tissue products, the risk is highly uncertain. That was reviewed by Peter
Bennet.
Touvin presented an independent risk
assessment from France that yielded virtually identical results as those reported
by Marc Turner. So, it is reassuring to
see two different risk assessment teams come up with very similar results.
For Australia, the Farrugia report,
they identified factor VIII as having the most risk among the human plasma
derived clotting factors.
Susie El Saadany stressed how
difficult it was to communicate these risks, as we have discussed heretofore,
due to all the uncertainties.
Johanne Lower from the Paul Erlich(?)
Institute, presented an extremely complex risk assessment, but there was also
-- I can't pretend to have understood the methodology, but I thought the
results were interesting.
They concluded that, under realistic
conditions, for Germany the disease should not become an endemic infection,
presuming that they get rid of the BSE. It should not become an endemic human
infection maintained by blood transfusions.
Transfused donors reduces the risk in
Germany only slightly, but the German conclusion should not be applied to other
countries that have different BSE and variant CJD risks.
Finally, as Dot Scott reviewed for you
today, due to uncertainties in the assumptions -- you will hear more about that
in the next meeting also -- it is very difficult to offer confident predictions
regarding the probability of variant CJD infections, not to mention illnesses
in people exposed to various blood components and plasma derivatives.
Let em close by going over the tables
of infectivity in human and animal tissues and other material. This was revised
from the previous table by a very energetic working group chaired by Paul
Brown, who is here today, with Gerald Hills and Ray Bradley handling the animal
tissue distribution.
I took the liberty of asking one of
the more artistic members of our group, Olga Maxiomva, to translate the table
-- it is not as colorful in this publication -- into a color tile table.
It is very difficult to see the whole
gestalt of those tissues that are either infectious or contain abnormal PRP and
those that don't and those that haven't been adequately tested, from a black
and white table.
So, we are presenting - and I divided
them up a little bit, just to make them easier for you to see. So, those issues in which infectivity or PRPTSE are detected I will present in red.
Those in which there is no detectable
infectivity or PRPTSE in some
reasonable number of experiments as judged by the working group I present in
dark blue.
Those that are either not tested, not
applicable or for which data are limited or are preliminary are in light blue.
When we come to the issue of blood,
with the exception of sheep with scrapie and BSE and humans with variant CJD,
the results were really quite controversial, contested, at the time of the
meeting.
It is hard not to be enthusiastic when
you see some of the results that were presented, and that is not in your
handout. You are going to see better data tomorrow, but I put it in this
presentation.
The preliminary data looked quite
good. On the other hand, the BSE community has been bitterly disappointed by
reports of detection by PLP TSE tests of agent in blood before, and there was a
certain reluctance to accept without further confirmation some of the
preliminary results on blood tests. Again, you will be seeing that in some
detail tomorrow.
So, high infectivity tissues are those
either in or anatomically associated with the nervous system. As you can see,
most of the tests with tissues clearly contain infectivity. As you know, a lot of that is in relatively
high titer.
Lower infectivity tissues, there are
also quite a few positives, although some of them are relatively consistently
negative.
One of the things that strikes me,
just from looking at the table, is that if you only saw part of this table, how
difficult it would be to predict from one animal model -- for instance, these
results from low infectivity tissues from sheep and goats, what you are going
to see in cattle.
It is interesting that the situation
in sheep nd goats seem to be more predictive of the variant CJD matter than was
the picture in cattle.
Going down the list, other lower
infectivity tissues, and here are the blood tests for PRPTSE tests for variant CJD or other forms of CJD,
BSE and scrapie in sheep and goats that we will be hearing about tomorrow.
I just want to show you what I mean by
the really exciting preliminary results that elicited two kinds of results, one
high enthusiasm, and the other concern that they looked almost too good to be
true.
This was the first test presented, and
you see that at least the claimed sensitivity overall -- that is, the number of
infected sheep, hamsters, cows, mice, monkeys and humans claimed was 100
percent of those tested, if we at least got the numbers down correctly.
Where there are missing data, it is
not because the developers that tested didn't know. It is because we somehow
didn't get it and the data wasn't provided when Jean Phillip was preparing the
summary in the same dose for the spike limit of detection.
The specificity also looks amazingly
high from the preliminary results. Only one false positive out of more than 100
bloods tested, five other tests, a total of six. Taken together they are not
quite as astounding as the first one. Still, overall, they look very good,
particularly the presented specificity.
Again, it is hard not to be
enthusiastic, but we have been burned before. So, the consultation felt that we
should really wait for confirmation of these results before accepting them as
bona fide detections of PRPTSE
in blood.
A number of issues, all attempts to
detect either infectivity or abnormal prion protein have been negative,
although I warn you, if you look on here you see an awful lot of light blue,
not tested, which shows you how much additional work would be needed before one
could be at all confident that there is an anti-infectivity in those tissues,
absolutely.
These are my own caveats. Limitations
of these studies come from the small numbers of human cases and animals
studied, the small numbers of bioassays attempted, the small volumes of
material sampled, the limits of detection usually unknown, some of the bioassay
animal species, as you saw, with outbred mice, with conventional mice with BSE.
Some of them are relatively prion sensitive.
The PRPTSE assays vary in sensitivity, some of them
relatively insensitive, very limited numbers of infected animals studied during
the incubation period.
Infected human beings almost never
identified and studied before the onset of overt disease. Not unimportant, the
uncertain relevance of one animal TSE model for predicting another animal TSE
distribution of infectivity, or the human distribution of infectivity.
I will close with the credits. The
chairman of the consultation was Pim van Aken from The Netherlands. Four of us
assembled the report with a great deal of help from those listed in the second
block down.
Paul Brown, Ray Bradley and Gerald
Wells worked very hard to revise these tables. Nothing is ever finished, and I
have an idea a couple of years from now we are going to see some revisions to
these tables. If there are any questions, I am happy to answer them. If not,
thank you.
DR. TELLING: Thanks, Dr. Asher. There is a question here.
DR. MANUELIDIS: What type of TSE tests were used in these
ones that look so spectacular?
DR. ASHER: You will hear all of them, from the actual --
that is why they are not in your handout. I think that the folks that developed
them ought to be able to present their own tests and their own data tomorrow.
We have a whole session on tests and
we invited everybody, every group, who had been represented at the WHO
consultation, because we don't like to play favorites.
We invited everyone to come, everyone
agreed to come, and we look forward to seeing what has gone on in the past
year, whether these things are continuing to develop in a promising way. Are we
getting close to a blood based screening test or is there more -- well, of
course there is more work to be done, but how close are we. You will have a
chance to judge for yourself tomorrow.
DR. TELLING: Thank you, Dr. Asher, very much for that
elegant summary. The final item on the agenda is the second open public
hearing. Dr. Freas, will you let us know who is registered for this open public
hearing session?
Agenda Item: Open Public Hearing.
DR. FREAS: Again, this is the time for members of the
public to address the committee on issues pending before the committee.
I have received two requests and they
will be called to the podium in the order in which I received the requests.
The first is Dr. Charles Sims, medical
director, California Cryobank, Incorporated. While Dr. Sims is approaching the
podium, our chair has to read the required and mandatory statement for the open
public hearing.
DR. TELLING: Both the Food and Drug Administration and the
public believe in a transparent process for information gathering and decision
making.
To assure such transparency at the
open public hearing session of the advisory committee meeting, FDA believes
that it is important to understand the context of an individual's presentation.
For this reason, FDA encourages you,
the open public hearing speaker, at the beginning of your written or oral
statement, to advise the committee of any financial relationship that you may
have with any company or any group that is likely to be impacted by the topic
of this meeting.
For example, the financial information
may include the company's or a group's payment of your travel, lodging or other
expenses in connection with your attendance at the meeting.
Likewise, FDA encourages you, at the
beginning of your statement, to advise the committee if you do not have such
financial relationships.
If you choose not to address this
issue or the financial relationships at the beginning of your statement, it
will not preclude you from speaking. So, Dr. Sims?
Agenda Item: Statement by Charles Sims.
DR. SIMS: I am Charles Sims, president of the
California Cryobank. By training and background, I am a pathologist, not what
many of you would consider a scientist, since most PhDs don't consider MDs
seriously in terms of science.
I have no expertise in prion disease.
I do have expertise in the field of reproductive medicine, in particular the
area of reproductive tissue banking.
I am the founder and the medical
director of one of the largest human sperm banks in the United States. I have
been active in this field since 1977.
The company paid for my trip here.
Since I am one of the principals of the company, it comes out of my pocket. I
have two conflicts of interest. One is that, as we are speaking at this moment,
I have an FDA inspection going on in my laboratory. I am not sure which is the
least pleasant of the two, but I appreciate being here.
My wife thinks that I spend too much
time traveling for business and not enough time traveling for pleasure. So,
those are my two conflicts of interest.
I am here to discuss and request that
this committee and the FDA reconsider the guidelines that are currently being
used for human sperm and egg donors.
The current guidelines were developed,
I believe, from everything I can read, to address the issues of blood and blood
products, not the issues of sperm or egg donors.
It is our opinion, in the field of
reproductive medicine, that those standards and those criteria and those
guidelines are not appropriate in this area.
Sperm donors is not a commodity like
blood. It is not a commodity like plasma products. It is not a commodity like
demineralized bone. Doctors don't choose
a sperm donor as a product.
The women themselves who are the
recipients of these are the ones who select the donor. They select an individual donor. They do not
select a sperm donor. It is a very important difference in those two.
To the best of my knowledge there has
been no transmission of CJD or any other type of transmissible spongiform
encephalopathy by sexual relations or activities.
When the guidelines by the FDA were
originally issued and I became aware of them, the applicability to reproductive
tissues -- namely egg donors and sperm donors -- at the time I thought that the
influence on that would be relatively trivial. I was wrong.
In 2006, when we applied the
guidelines to reproductive tissue donors, sperm donors in particular, we lost
20 donors out of our program. That amounted to about 13 percent of the active
donors in our program. That is a material effect.
It may seem trivial to the rest of you
but, to us, it is not trivial. It is very, very difficult to recruit sperm
donors.
There has been a lot of negative
publicity and media about donors losing their anonymity, and it is becoming
increasingly difficult to attract and retain donors.
A blood donor can simply walk into the
blood donor center, take a short history and physical, check the hemoglobin,
and then they can accept it as a donor, and that is the end of the story.
A sperm donor, it takes weeks and
sometimes a few months to fully qualify as a sperm donor. It is a long
elaborate process. We know more about our sperm donors than I would say most of
you knew about your spouses when you got married.
We do a three generation medical and
genetic history, we do extensive testing over a period of time. We wind up, out
of 100 donors who apply to become donors, only two or three actually wind up
being in our final donor pool.
So, we lose something like 97 or 98
percent of the potential applicants, the potential applicants. We lose them for
a large number of reasons. The single largest reason is probably poor sperm
production.
The fertility parameters are
suboptimal for our purposes. They are normal for normal reproduction, but they
are suboptimal for sperm banks. Because of the freezing and thawing, we lose
about 50 percent of the motile cells when they go through that process. So, we need to start out with higher
reproductive parameters than average. We lose them for many reasons.
Each one of these small barriers,
which may seem trivial, they all add up. The cumulative effect of that is that
it is getting harder and harder and harder.
I am the immediate past chair of the
reproductive council of the American Association of Tissue Banks. I just
conducted a national survey of all the sperm banks in the United States that
provide donor semen. So, I am very familiar with the issues that the directors
of those programs have.
Universally, they tell me that the
single largest problem they have is finding and keeping sperm donors. It is not
an easy task.
To have additional barriers placed on
us, such as the exclusion of donors who lived in the United Kingdom for six
months or three months or a military base in Greece for six months or some place
else in the United Kingdom for five years seems trivial, I am sure, to many of
you but to me it is not trivial.
It has bene painful, it is hard. It is
hard to replace these donors. I think we may have had more than the usual
number of donors of that, because of the places that our laboratories are
located. They are in cities with a more
cosmopolitan and perhaps more widely traveled group.
I would like to explain and give you
just a little context about sperm banks and sperm donors and what this goes
through.
First of all, I would like to address
the issue of processing sperm. We don't pool sperm. You pool blood, you pool a
lot of things, but sperm is never pooled. Egg donors are never pooled. So, all
of these are very individual things.
Nearly all the inseminations done in
the United States are done with the intrauterine method. The intrauterine
method achieves about twice the fertility rate per cycle that intracervical
inseminations do. That is the reason why nearly all specialists in this area have
moved from cervical insemination to intrauterine insemination.
In order to do the intrauterine
insemination, you have to remove the seminal plasma. The reason you have to
remove the seminal plasma is because seminal plasma contain prostaglandins,
causes uterine contractions.
So, they can be trivial or they can be
more severe. So, that is universally done. Most and much of the sperm washing
that is done to remove the seminal plasma also removes leukocytes.
If you use density gradient methods
you remove nearly all of the leukocytes as well as the seminal plasma. You also
get rid of the non-motile fraction of sperm to a large extent using the density
gradient methods.
If you look at the biomass or the
bioburden that donor semen provides, it is about -- I would estimate that it is
somewhere between an eighth to a tenth of what a normal human ejaculate has.
The normal human ejaculate has three
to four ml of volume and we take that, we remove the seminal plasma, and we
re-suspend the density gradient cells, in our case, into a sperm media that has
cryoprotectant and other buffers in it.
That winds up being only about a tenth
or so of the original volume of ejaculate.
In normal sexual relationships, the exposure or bioburden is nearly 10
times that.
There is far more normal sexual
activity that goes on in the country and the world than there are sperm
donations. There have been no reports, to my knowledge, of the transmission of
any form of CJD by sexual transmission between spouses or others, and there has
been none as far as donors are concerned.
It is for this reason that we feel
that the use of the blood donor criteria is inappropriate for sperm banks. My
friend, David Mortimer, published a paper in Reproductive Medicine on line just
this past week, which you have in your handout, which lays out basically the
basic arguments for asking for this exemption from the blood products and blood
guidelines.
I would be happy to answer any
questions you may have about sperm banking. I am not capable of answering detailed
questions about prions.
DR. TELLING: Thank you, Dr. Sims. I
will entertain questions with regard to points of clarification, if there are
any right now.
If there are none, thank you once
again, and I would ask Dr. Brown, representing Nordisk Cryobank, to come and
speak to us.
Agenda Item: Statement by Paul Brown.
DR. BROWN: Thank you very much, Glenn. It is a little
disorienting to speaking to the group from here. I used to speak from there and
be the judge.
A month or so ago I got an e mail from
a Danish company. As you know now, it is Nordisk Cryobank, and they wanted me
to talk to you today.
I sent them a message back saying,
well what do you want me to talk about. They said, well, we would like you to
talk about restrictions on importing our product.
What is that? Well, the product is sperm. I said, well, I
don't really have anything much to do with that. Besides, I said, the meeting
that you are going to is basically all about plasma. It is really not concentrating
on tissues or cells.
They said, well, what about the WHO
guidelines? ? That seems to be relevant. I thought about it for a little while.
I decided, like an attorney accepting
a case to prove a principle in front of the Supreme Court, that I would take on
the request. So, I have, in fact, come to you at the best and in the employ as
a consult of the Danish Cryobank that historically has contributed sperm donations
to the United States.
In the context of full disclosure, I
should probably mention to you three unhappy facts. The first is, I have never
made a donation myself.
The second, worse, I have never been
invited to make a donation. The worst of all is, my banking days are long past.
So, with that preamble, and to make this as painless as possible, what I am
going to do really is just read into the record the handout that you have in
front of you.
In June 2002, which you have heard,
the FDA published a draft guidance on human cells, tissues and cellular and
tissue based products, in relation to the risk of contracting variant
Creutzfeldt Jakob disease. This guidance has still not been finalized.
In September of last year, it should
be actually, not 2006 but 2005, the WHO held their consultation that Dave has
just summarized for you. It included the
table that Dave also has referred to.
In point of fact, the risk from
variant Creutzfeldt Jakob disease tissue is a function of several things.
It is a function, like any other
infectious disease, transmissible disease, it is a function of the geographic
location of the donors, to what they may have been exposed, the level of
infectivity in the tissue, obviously, any reduction of that infectivity that
will be the result of processing.
It also has to do with the route of
administration. We all know that if you give something that has CJD agents in
it directly into the brain you will do a good deal more damage than if you are
giving it from any number of peripheral routes, and finally, it is a function
of dose.
You know from work on blood that you
can transfuse an infection into a sheep using 200 ml of plasma or whole blood
intravenously far more effectively than you can by inoculating 0.03 ml of buffy
coat into the brain directly.
So, all of these factors have to be
taken into consideration in a risk assessment. The example that I have been
asked to present to you is the excluded tissue of sperm.
The guidance that is still in draft
form from 2002 recommends the exclusion -- actually really only addresses one
of those factors and it is the geographic source.
They recommend, as you well know, the
exclusion of donors with a total residence time since 1980 of three months or
more in the United Kingdom and five or more years in all countries in
continental Europe.
One consequence of this is the
exclusion of all cells, tissues and tissue products from the entire European
native population, obviously.
So, looking at the five factors to see
how realistic this exclusion is, Denmark has had a thorough systematic active
surveillance of BSE for seven years and CJD for 10.
During this time, there have been a
total of 14 cases of BSE, decreasing numbers each year, and this year none at
all.
Around 54 cases of CJD that have been
identified, none have had the variant form of the disease. As a matter of fact,
outside of France, there have been only five cases of variant CJD identified in
the entirety of continental Europe with a population on the order of something
like 300 million people.
So, it is really no longer sensible to
group France and the rest of Europe in a single basket. It is also worth noting
that, during the same period, Canada has had nine cases of BSE including one in
2006, no cases of variant, as in Denmark, but the FDA has not thought it
necessary to restrict donations from the Canadian population.
The second point, tissue infectivity
levels. The WHO panel did construct a table and showed that all experimental
attempts to transmit disease from reproductive tissues, including semen, from
cattle with BSE and from sheep with scrapie have failed.
These have been substantial
experiments. Limited experiments in humans have also failed. More expensive
examination of the pathologic prion protein have been done and have been
uniformly negative, both in animals and in humans.
I think even stronger support for the
notion that semen and sperm in particular is not transmissible comes from the
epidemiologic observation that there has not been, despite decades of
searching, a single proven instance of vertical transmission.
The USDA, interestingly enough,
permits the importation of bovine sperm even from the United Kingdom, if
several criteria are met that minimize the possibility of an infected
donor. Those criteria do not include a
bovine travel history.
The third point, tissue processing,
you have just heard and I just summarize or recapitulate the processing of
semen into sperm, both in Denmark and in the bank that you have just heard
about, involves a density gradient which reduces leukocytes from one to two
million maximum per milliliter, to undetectable levels of 10,000 cells or less
per ml. That is below the threshold that is acceptable for leukodepleted blood.
In addition, after it is washed, and
as you probably have heard from Bob Rohrer, there is some evidence that simple
washing will reduce the amount of infectious agent attached to cells in the
blood.
The fourth point, route of
administration. Venereal transmission of disease has not been adequately
studied experimentally, but epidemiologic evidence indicates that it does not
occur.
Finally, the total amount of
administered tissue, as you have just heard, is about a tenth of a single
ejaculation, about a half an ml in volume.
It is important to know that you can
actually have tissue that is infectious and give it, as I mentioned earlier, in
a dose that is too small actually to be transmitted.
That can be overcome. In a very nice
paper a year or so ago, that can be overcome by administering repeated
subtransmissible doses, but insemination is a one shot procedure.
So, a consideration, it seems to me,
of all of this information leads to the conclusion that sperm donated from
healthy, young individuals living in Denmark does not pose a risk of variant
CJD transmission.
Equally important and more general,
the FDA donor exclusion criteria really do need to be revised to reflect both
the change in BSE vCJD situation in Europe and the most recent information on
tissue infectivity. Thank you very much.
DR. TELLING: Thanks, Dr. Brown. I will once again
entertain questions related to clarification.
DR. MANUELIDIS: Actually, I sort of agree with that. I think
that it is important to take cells, cells of the body, et cetera, as a very
separate type of issue from blood.
There are many types of cells. Just to
be equal about it -- as Paul knows, I like to be equal -- we haven't considered
egg cells either.
Actually, there is one thing that
confuses me. We did lots of experiments with guinea pigs, with insemination,
and we kept them for 12 years, and not a single case of insemination or
cohabitation with CJD infected guinea pigs with each other led to any kind of
infection.
However, in 1989 -- maybe you have a
follow up for this -- when I was in the United Kingdom and they were talking
about the spread of BSE, it turns out that they had sent -- they felt that the
source of the BSE in Saudi Arabia, they had sent a breeding bull to Saudi
Arabia, and that is what they felt was the source of the Saudi Arabian BSE
cases.
So, maybe you could follow up on that.
I think if that were ruled out, that would also be a very interesting point.
DR. TELliNg: I am just going to make this point. Because
this is an issue that is not on the agenda, we are not really at liberty to
discuss it in any depth today.
I did want to give you the opportunity
for questions of clarification. If there is a question of clarification, then I
will entertain it, but otherwise maybe we should move on.
DR. MANUELIDIS: I just wanted a clarification about the case
that was sent to Saudi Arabia.
DR. SIMS: I have no clarification, and
I think any American effort to get clarification from Saudi Arabia, at this
point in time, will fail. Perhaps in the future it will succeed.
The reason that this presentation has
been made, at least from my point of view, is that I do think it is a good case
study in why you are going to have to re-address, we are not going to come back
to a consideration of excluding European tissues and cells on the basis of
information.
The anchor to this meeting was today's
discussion. If you have no evidence of infectivity in the tissue, it just
strikes me as unreasonable to group it in with tissues for which you do have
evidence.
DR. TELLING: And the FDA is happy to listen to these
comments.
DR. SALMAN: This is to clarify about the Saudi Arabia
semen, which was rejected, that hypothesis. The main source for that infection
was the importation of the MBM from the United Kingdom.
DR. TELLING: Thank you, Dr. Brown, and thank you for your
full and frank disclosure also. So, with
that, I would like to thank everybody once again.
DR. FREAS: I do have two quick announcements. [Housekeeping/logistics matters discussed.]
DR. TELLING: So, the meeting is adjourned. Thank you very
much.
[Whereupon, at 3:55 p.m., the meeting
was recessed, to reconvene the following day, Tuesday, September 19, 2006.}